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Developmental Biology 233, 347–364 (2001) doi:10.1006/dbio.2001.0227, available online at http://www.idealibrary.com on Study of the Murine by Allantoic Explants

Karen M. Downs,1 Roselynn Temkin, Shannon Gifford, and Jacalyn McHugh Department of Anatomy, University of Wisconsin–Madison Medical School, 1300 University Avenue, Madison, Wisconsin 53706

The murine allantois will become the and vein of the chorioallantoic . In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos. In this study, the extent to which explanted allantoises grow and differentiate outside of the was investigated. The explant model was then used to elucidate cell and growth factor requirements in allantoic development. Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes. Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises. DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent. Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF164. Although VEGF164 was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelial- ization of these cells. Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles. On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems. © 2001 Academic Press Key Words: allantois; angioblasts; ; embryos; endothelium; flk1; flt1; immunohistochemistry; in vitro; lacZ; mesenchyme; mesothelium; mouse; placenta; smooth muscle; tie1; tie2; transplantation; vasculogenesis; VCAM1; VEGF.

INTRODUCTION The allantois will differentiate into the umbilical com- ponent of the placenta and thus produces one of three major Eutherian rely upon formation of a placenta to circulatory systems in the conceptus. Until recently, few mediate exchange of nutrients, wastes, and gases with the definitive studies have focused on the allantois. Little was mother during gestation. The process of placentation pre- known about it other than what it looked like (e.g., Elling- sents a dramatic example of cooperation between two ton, 1985) and from where it originated in the conceptus genetically distinct organisms sharing a common physiol- (Gardner et al., 1985; Lawson et al., 1991). ogy. Mouse placentation, like that of humans and primates, The murine allantois first appears during early gastrula- involves at least three pivotal events: formation of the tion as a small bud of extraembryonic (Jolly and allantois and the chorion, chorioallantoic union, and vas- Fe´rester-Tadie´, 1936; Bonnevie, 1950; Snell and Stevens, cularization. The sequence and fidelity of these morphoge- 1966; Kaufman, 1992) that is derived from proximal epiblast netic processes are critical to the survival and health of the (Gardner et al., 1985; Lawson et al., 1991). The absence of fetus, yet many of their most fundamental aspects are still endoderm in the allantois has been suggested by electron mysterious, with few clear precedents concerning cell and microscopy in rats (Ellington, 1985) as well as by fate molecular requirements. mapping studies which ruled out the contribution of primi- tive endoderm-derived cells (Gardner and Cockroft, 1998). 1 To whom correspondence should be addressed. Fax: (608) 262- Between the late neural plate and 6-somite-pair stages 7306. E-mail: [email protected]. (approximately 7.75 days postcoitum, dpc, through 8.5 dpc),

0012-1606/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 347 348 Downs et al. the allantoic bud lengthens by several mechanisms. These MATERIALS AND METHODS include the addition of mesoderm from the primitive streak (Tam and Beddington, 1987; Lawson et al., 1991; Downs Mouse Strains and Bertler, 2000), mitosis (Ellington, 1985; Downs and Bertler, 2000), and distal cavitation (Ellington, 1985; Brown Two mouse strains provided embryonic material for this study and Papaioannou, 1993). and were maintained on a 12-h light/dark cycle (dark period As the allantois grows in the exocoelom, it acquires an 13:00–1:00): (1) matings between the F1 generation of (C57BL/6 ϫ outer sheath of mesothelium and an inner core of vascular- CBA) provided standard embryonic material and (2) F1 females of ϫ izing mesoderm. Despite the absence of endoderm, gener- the (C57BL/6 CBA) strain mated with homozygous lacZ/lacZ ally held to be required for de novo formation of blood males of similar genetic background (ROSA26*; Downs and Har- mann, 1997) provided lacZ-expressing donor allantoises for trans- vessels (Risau and Flamme, 1995), the murine allantois plantation. vascularizes initially by vasculogenesis, rather than by invasion from the vitelline or fetal vasculatures (Downs et al., 1998). Allantoic vasculogenesis is not accompanied by Dissection, Staging, and Culture of erythropoiesis. Differentiation of allantoic mesoderm is directional, beginning distally and continuing proximally Pregnant females were killed by cervical dislocation at 11:00 AM (Downs and Harmann, 1997; Downs et al., 1998). Between on the 8th day of gestation (approximately 7.75 dpc) or the 4 and 6 somite pairs, allantoic mesothelium mediates following day at the same time (approximately 8.75 dpc). Concep- chorioallantoic union (Downs and Gardner, 1995; reviewed tuses were dissected from implantation sites into Dulbecco’s in Downs, 1998). By about 10 somite pairs, allantoises are modified Eagle’s medium (DMEM)-based dissection medium (Law- overtly vascularized and contain large numbers of primitive son et al., 1986; Downs and Gardner, 1995), Reichert’s membrane and associated were reflected, and embryos were staged erythrocytes. By approximately 11 dpc, a single artery and as described previously (Downs and Davies, 1993) and in Brown vein have formed. (1990). Normal headfold-stage conceptuses were cultured in 1 ml of Visual analysis and experimental manipulation of the DMEM-based culture medium containing immediately centrifuged allantois in whole embryo culture have been essential for and heat-inactivated rat serum (1:1; Lawson et al., 1986; Downs discovering the developmental mechanisms and spatiotem- and Gardner, 1995) for up to 24 h if they were to be used for poral features of allantoic growth and differentiation. None- immunohistochemistry or for up to 32 h if they were to be used as theless, use of the whole conceptus to study allantoic hosts and control conceptuses in transplantation experiments. development is limited. Ideally, manipulation of allan- Conceptuses were scored as previously described (Downs and ϭ toises in isolation would be extremely useful to elucidate, Gardner, 1995); 1.7% of conceptuses (n 116) with stunted for example, the emergence and coordinated formation of growth of the allantois were excluded from the study. allantoic cell lineages and to investigate the effect of varying concentrations of growth factors on allantoic vas- cularization. Results of previous studies have suggested Culture of Allantoises and DiI-Labeling that the allantois may be ideally suited to development Allantoises were mouth-aspirated into a hand-pulled glass mi- outside of the conceptus, as isolated allantoic tips matured crocapillary (Downs and Gardner, 1995; Downs et al., 1998; Downs and fused with the chorion at the appropriate time, despite and Bertler, 2000) and either cultured in suspension (Downs et al., lack of connection with the fetus (Downs and Gardner, 1998) or placed individually on plastic or poly-D-lysine-coated 1995). Moreover, formation of the nascent allantoic vascu- (Sigma; 1 mg/ml double-processed tissue culture water, filtered lature appears to be self-contained, relying upon de novo through 0.45-␮m, cellulose acetate, coated for 30 min, and rinsed conversion of allantoic mesoderm into the endothelial cell 4–5 times with sterile double-processed water) glass coverslips (No. 1 thickness, 12 mm; Fisher) inserted into 24-well tissue- lineage (Downs et al., 1998). Also, allantoises explanted to culture plates (Falcon 3047). Explants were cultured in 0.5 ml of tissue culture plastic contained a widespread Flk-1-containing culture medium containing 50% rat serum (“high serum,” Downs vascular plexus at the end of 24 h (Downs et al., 1998). Thus, and Gardner, 1995) or 5% (“low serum”) or 50% fetal calf serum if allantoic development could be recapitulated outside of the (FCS; Gibco BRL; frozen and thawed twice before using). For conceptus, use of explanted allantoises may shed light on culture longer than 1 day, explants were given completely fresh mechanisms controlling early placentation. gas-equilibrated medium at 24-h intervals. To test the allantoic explant model and use it to elucidate Labeling the allantois was carried out in some experiments by the molecular and cellular requirements of allantoic devel- dipping whole allantoises, distal tips, or the proximal (basal) region opment, we have combined tissue culture, immunohisto- of the allantois into a solution of DiI (DiI/DiIC18(3); Molecular chemistry, transplantation, and growth factor analysis in Probes; 1 part 0.5% DiI in absolute alcohol:9 parts 0.3 M sucrose) as the allantois was held by mouth aspiration in a capillary of similar explants of early headfold-stage allantoises. Our results diameter. Immediately after culture on poly-D-lysine coated glass provide strong evidence that the murine allantois, both coverslips, labeled explants were fixed in 4% paraformaldehyde for intact and in isolation, is a unique and highly promising 1 h. They were then rinsed in PBS and then double-processed water, new tool to discover how mesoderm is transformed into the mounted in glycerol-based mounting medium on glass slides, and endothelial cell lineage of the umbilicus and its supporting examined in the compound microscope with rhodamine excitation cells. (G2-A filter cube, excitation 535/50; emission 590).

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. In Vitro Vasculogenesis in the Mouse Allantois 349

Histology and Immunohistochemistry Gibco BRL) at 37°C in 6.2% CO2 for 10 min. Dissection medium (0.5 ml) inactivated the trypsin, after which explants were tritu- Experiments involving immunohistochemistry were carried out rated 10 times, pooled, and centrifuged at approximately 1000 rpm minimally in triplicate for every time point, treatment, or devel- for 15 min. The cell pellet was resuspended in DMEM-based opmental stage (Ն3 conceptuses per experiment) and included dissection medium (Downs and Gardner, 1995). In preliminary appropriate controls (absence of antibody, prebinding with control experiments, dissociated cells were counted with a hemacytometer peptides; Downs et al., 1998). and the proportion of single and clumped cells was noted (legend to Immunohistochemistry on explants and paraffin-embedded sec- Table 2). In some experiments, dissociated cells of lacZ/ϩ genotype tions was carried out as previously described (Downs et al., 1998), were replated to verify that all of them retained lacZ expression. with the following exceptions: (i) plated explants were cultured in P ϭ 24-well tissue culture dishes and (ii) immunohistochemistry (all No significant differences ( 0.77) were found in cell numbers ϩ ϩ Ϯ ϭ antibodies and cognate control peptides were from Santa Cruz between / (five experiments, 5101 432 cells per allantois, n ϩ Ϯ Biotechnology, Inc.: Flk-1, SC-315; Flt-1, SC-316; Tie-1, SC-342; 29; Table 1) and lacZ/ (three experiments, 5388 1046 cells per ϭ Tie-2, SC-324; VCAM-1, SC-1504, SC-8304; VEGF, SC-1836) in allantois, n 31; footnote in Table 1) explants cultured for 24 h in paraffin-embedded tissue included an unmasking step in the mi- high serum. crowave according to the suppliers. After the last heat cycle, To verify that all explanted cell types expressed lacZ, some sections were placed in PBS in preparation for immunohistochem- lacZ/ϩ explants were stained with X-gal (Downs and Harmann, istry. The chromogen diaminobenzidine (DAB; Sigma) or metal- 1997) at 37°C for 12–15 h after fixation in 4% paraformaldehyde at enhanced DAB (Pierce) was used to detect binding of horseradish 4°C for 30 min. Longer fixation times (i.e., 90 min) blocked X-gal peroxidase-conjugated secondary antibodies in single-antibody ex- staining in isolated outlying cells, which were spread out more periments. In experiments involving use of two antibodies, alkaline thinly on the plate. phosphatase (AP) was conjugated to the secondary antibody and For transplantation, 1 ␮l of the donor cell suspension was Fast Blue BB salts (Sigma) were used to detect AP activity (Miller, mouth-aspirated into a thin hand-pulled glass capillary (i.d. ap- 1996). proximately 40 ␮m), which was then used to inject the donor cells into headfold-stage conceptuses that had been cultured for 24 h Dissociation and Transplantation of Cultured (Downs and Gardner, 1995). Two hosts were judged abnormal (allantois not fused with chorion) and were discarded. After injec- Allantoic Cells tion, 28 operated and control conceptuses were cultured individu- Plated allantoises were dissociated in 0.5 ml of trypsin solution ally for a further8hinfresh culture medium, after which all (0.05% trypsin, 0.53 mM EDTA in Hanks’ balanced salt solution; conceptuses contained approximately 14–16 somite pairs. All

TABLE 1 Cell Numbers in Allantoic Explants

Conc. of VEGF Exogenous Average cell %of (pg/ml) Ϯ SEM

VEGF164 Culture No. of No. of No. per allantois normal after culture Growth condition (ng/ml) time (h) experiments allantoises (ϮSEM) levels (No. of allantoises)

50% rat serum 0 24 5 29 5101a (Ϯ432) 100 35.5 Ϯ 0.6b (6) 5% fetal calf serum 0 3 13 4556 (Ϯ481) 89.3 50% rat serum 0 72 14 52 6516 (Ϯ723) 100 27.5 Ϯ 0.5b (6) 10 3 9 6429 (Ϯ257) 98.7 50 6 27 8171 (Ϯ1706) 125.4 5% fetal calf serum 0 9 26 3637 (Ϯ679) 55.8 0.02 5 9 4062 (Ϯ171) 62.3 2 4 11 5242 (Ϯ198) 80.4 10 5 14 5918 (Ϯ193) 90.8 50 3 9 5376 (Ϯ806) 82.5

Note. All experiments described here were carried out minimally in triplicate. Early headfold-stage allantoises of ϩ/ϩ genotype were placed onto tissue culture plastic in 0.5 ml of culture medium. Concentrations of VEGF were determined by ELISA (see Materials and Methods). No difference was found between unincubated “fresh” rat serum (52.0 Ϯ 5.1 pg/ml (n ϭ 4 samples)) and rat serum incubated for

24 h at 37°C in 6.2% CO2 (54.2 Ϯ 3.3 pg/ml (n ϭ 14 samples)). The concentration of VEGF in “high” serum after 24 h incubation was 34.1 Ϯ 3.0 pg/ml (n ϭ 8 samples), more than the predicted 27.1 pg/ml and possibly due to inaccuracies introduced as a result of spinning down and diluting the serum before use. Pipetting accuracy was calculated immediately after dispensing 50 ng/ml recombinant VEGF164 to high-serum culture media and found to be 51.5 Ϯ 1.9 ng/ml (n ϭ 15 samples). Thereafter, exogenous VEGF was added to culture media without subsequent ELISA measurements. a The average number of cells per headfold-stage explant of lacZ/ϩ genotype cultured for 24 h in 50% rat serum was 5388 (Ϯ1046; 3 experiments, n ϭ 31) and was not significantly different from ϩ/ϩ explants (P ϭ 0.77). b The concentrations of VEGF in 24- and 72-h high-serum explants were not significantly (P Ͼ 0.05) different, as each time point was carried out as a separate experiment internally controlled with high-serum media that did not contain explants.

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FIG. 1. Localization of Flk-1 in 24- and 72-h allantoises explanted to high serum. Bright-field micrographs of headfold-stage allantoises explanted to high-serum (50% rat serum) culture medium in 24-well tissue culture plates and immunostained for Flk-1 (brown color). (A) 24-h explant shows the extensive Flk-1-positive vasculature. The boxed area is enlarged in (B). (B) Boxed region of (A) shows individual Flk-1-positive cells (arrowheads) on mesenchyme at the periphery of the explant. (C) 72-h explant shows a reduction in the Flk-1-positive vasculature with small clusters of Flk-1-positive cells (arrowheads) and relatively uniform widening of vessel lumina. (D) Individual and small clusters of Flk-1-positive cells (arrowheads) on mesenchyme (arrows) found at the periphery of 72-h allantoic explants. Abbreviations: e, endothelial cell lineage; m, mesenchyme. Scale bar in (D): 200 ␮m (A, C), 400 ␮m (B), 800 ␮m (D).

conceptuses were fixed and processed for X-gal staining as de- mean (SEM). In Table 1, the Student’s two-tailed t test (Mini-Tab, scribed previously (Downs and Harmann, 1997). equal variances assumed, confidence interval 95.0%, “pooled” subcommand) determined significant differences (P Ͻ 0.05) in concentrations of VEGF in the culture medium of 24- and 72-h Quantitative Assay for Vascular Endothelial explants and between cell numbers in 24-h allantoic explants of Growth Factor (VEGF) ϩ/ϩ and lacZ/ϩ genotype.

Recombinant vascular endothelial growth factor (VEGF164; R&D Systems, Minneapolis, MN) was prepared according to the manu- RESULTS facturer’s instructions. Concentrations of mouse and rat VEGF were determined by a quantitative sandwich enzyme immunoassay Growth and Morphology of Allantoic Explants technique, according to the manufacturer’s instructions (Quan- at 24 and 72 h tikine M Murine; R&D Systems), which recognized three VEGF isoforms, 120, 164, and 188, of mouse and rat, but not bovine Early headfold-stage allantoises were removed from wild- VEGF. type conceptuses, introduced individually into plastic wells containing high serum, and examined at 24-h intervals for up to 3 days. Early headfold-stage allantoises were selected Statistical Analyses because they are large enough to manipulate and relatively Mini-Tab (Mini-Tab, Inc., State College, PA) was used through- undifferentiated. At this stage, about 35% of allantoic cells out this study to calculate the average and standard errors of the contain Flk-1, most of which localize to the distal two-

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FIG. 2. Mean diameter (in ␮m) of fed and unfed allantoic explants. Single allantoises were plated directly onto plastic and cultured for 24, 48, or 72 h. “Fed” cultures were given complete high serum media replenishment at 24-h intervals, whereas “Unfed” cultures were not. Numbers in the base of each column represent the number of explants scored. The error bars on top of each column represent SEMs.

thirds of the allantois and have not yet undergone signifi- (Fig. 5, and data not shown). Thus, allantoic explants both cant morphological differentiation (Downs et al., 1998). proliferated and differentiated in vitro. High-serum medium was selected because it had been used In the absence of feeding, vascular channels began to extensively in previous studies (Downs and Gardner, 1995; break down at 48 h such that, by 72 h, only 18.2% of the Downs and Harmann, 1995; Downs et al., 1998; Downs and unfed cultures retained vasculature (Fig. 2 and data not Bertler, 2000). shown); the remaining cultures contained attached cells We had shown previously that 24-h allantoic explants resembling mesenchyme. Feeding cultures at 24 and 48 h vascularized and expressed Flk-1 (Fig. 1A; Downs et al., resulted in 100% explant survival and more cells by 72 h 1998). We show here that, over a 24-h period, explants (Fig. 2, Table 1, and data not shown). Although the vascu- enlarged approximately seven times (Table 1) compared lature was intact and robust in the majority of explants, it with cell numbers in allantoises at the headfold stage was generally less extensive than observed at the previous (Downs and Bertler, 2000). Also, when plated onto poly-D- two time points (Fig. 1C). At all time points, small clusters lysine-coated glass coverslips, all explants formed a robust of Flk-1-containing angioblasts were found on top of at- capillary network consisting of vessels of varying thickness, tached mesenchymal cells, the latter of which did not the only perceptible difference being that vascularization express flk1 (Figs. 1B and 1D). No attempt was made to on glass lagged approximately 4–5 h behind that on plastic maintain the explants beyond three days. Thus, not only

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FIG. 3. Time course of phenotypic changes in explanted headfold-stage allantoises. Headfold-stage allantoises were removed from conceptuses (n ϭ 5 experiments, Ն3 allantoises per time point per experiment), plated in 24-well tissue culture plates containing high serum, and examined at the following time points: (A) 4 h, (B) 8 h, (C) 12 h, (D) 16 h, and (E) 20 h. Straight black arrows (C–E) point to vascular channels; white arrows (D, E) point out blebs of clustered cells of the endothelial cell lineage (described in Downs et al., 1998, and in Fig. 1, this study); curved arrows (B–E) point to mesenchymal cells; asterisk in (A–D) marks the proximal region. Scale bar in (E): 100 ␮m (A), 240 ␮m (B, D, E), 250 ␮m (C). did high serum with daily replenishment sustain allantoic At 4 h into culture, allantoises had attached to the plastic cell proliferation and differentiation, but it promoted vas- but had changed little from their initial gross morphology cular remodeling. (Fig. 3A). At 8 h, most explants were well attached to the plastic and exhibited mesenchymal cells spreading out from the distal region of the allantois (Fig. 3B). By 12 h, all Time Course of Vascularization in Explanted allantoises were well adhered to the plastic and most were Allantoises still somewhat elongated. At one end, cells were beginning To discover whether isolated allantoises in culture ad- to vascularize, whereas at the other, cells appeared morpho- hered to a strict developmental program, a 24-h time course logically undifferentiated (Fig. 3C). DiI labeling (Figs. 4A– was carried out at 4-h intervals. Also, to determine whether 4G) proved that the latter contained the base of the allantois the directionality of vascularization was maintained in whose cells eventually became incorporated into the endo- culture, the proximal and distal ends of some allantoises thelial, mesothelial, and attached mesenchymal popula- were labeled with DiI prior to plating. tions (described in the next section). Although dispersed,

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of isolation, allantoises adhere to a stereotypic program of differentiation and that polarity of differentiation is main- tained with little cell mixing.

Gene Expression in Allantoic Explants Identifies Endothelium, Mesothelium, and Mesenchyme Endothelial cell lineage. Immunohistochemistry aided in the identification of cell types in allantoic explants. Intact allantoises expressed four genetic markers of the early endothelial cell lineage, flk1, flt1, tie1, and tie2 (Shalaby et al., 1995; Fong et al., 1995; Sato et al., 1995) with no perceptible differences between freshly recovered and cultured conceptuses of approximately equivalent de- velopmental stage (9–16 somite pairs, approximately 9.0 dpc; Figs. 5A, 5B, 5D, 5G, 5H, and 5J and data not shown). Expression of each of the four genes was found within the base of the allantois, presumably in putative angioblasts and nascent blood vessels (example shown in Fig. 5G). However, levels of flt1, tie1, and tie2Јs gene products were more heterogeneous in the endothelial cell lineage than those of flk1, which were generally high in both angioblasts and vascular channels. Moreover, each of the four gene products was found in derivatives of trophectoderm (here- after referred to collectively as “trophoblast”), including the chorionic ectoderm, ectoplacental cone, and many tropho- blastic giant cells (e.g., Fig. 5H). To confirm that expression was intrinsic to trophoblast and not to penetration of the FIG. 4. DiI labeling of the proximal and distal ends of whole chorionic disk by allantoic endothelial cells, whole concep- allantoises. The proximal or distal ends of whole allantoises were tuses underwent microsurgical removal of prefusion allan- labeled with DiI (see Materials and Methods). Explants were toises (Downs and Gardner, 1995), 24-h culture, histological cultured for 24–48 h but examined at 12-h intervals to monitor the preparation, and immunostaining. Where allantoic regener- label relative to the clump of morphologically obscure cells in Figs. ates had failed to reach the chorion, trophoblast nonethe- 3C and 3D. (A) Phase-contrast and (B) fluorescence photomicro- less contained Flk-1, Flt-1, Tie-1, and Tie-2 (data not graphs showing DiI labeling of the proximal end of a headfold-stage shown). allantois. (C) Superimposed phase-contrast/fluorescence photomi- Flk-1, Flt-1, Tie-1, and Tie-2 were examined in 24-h crograph of the explant in (A, B) after 48 h in culture. (D) explants (Figs. 1A, 1B, 5C, 5E, 5F, 5I, 5K, and 5L). Flk-1 was Phase-contrast and (E) fluorescence photomicrographs showing DiI labeling of the distal end of a headfold-stage allantois. (F) Phase- abundant in angioblasts and endothelial cells (Figs. 1 and 5), contrast and (G) bright-field photomicrographs showing the ex- whereas anti-Flt-1, Tie-1, and Tie-2 intensely stained angio- plant in (D, E) 24 h later. Asterisks indicate the proximal allantoic blasts and most, but not all, of the vasculature (Figs. 5E, 5F, region in all photographs, and the black line in (C) delineates the 5I, 5K, and 5L and data not shown). Mitotic figures were proximal end of the explant. Arrows point to regions containing easily identified in cells containing each of the four gene DiI-labeled cells. DiI labeling of the whole allantois or tip alone had products (data not shown). In 72-h explants, levels of Flk-1, no effect on morphological differentiation or its polarity (data not Flt-1, and Tie-2 remained unchanged (Fig. 1C and data not ␮ ␮ shown). Scale bar in (G): 200 m (A, B, C, D, E), 100 m (F, G). shown); Tie-1 was present but reduced (data not shown). Mesothelium. In intact allantoises, mesothelium nor- mally envelops the vascularizing allantoic core; its only known function is to mediate allantoic attachment to the labeled cells remained segregated at their site of labeling chorion (Downs and Gardner, 1995) via molecular interac- (Figs. 4C and 4G). At 16 h, crisscrossing vascular channels tions between VCAM-1 and ␣4-integrin (Gurtner et al., almost fully obscured the few cells still piled up at the 1995; Kwee et al., 1995; Yang et al., 1995). Intriguingly, we proximal end of the allantois (Fig. 3D). Allantoises cultured found that VCAM-1 was localized to the distal two-thirds of for 20 h (Fig. 3E) were indistinguishable from those at 24 h, the mesothelium of intact allantoises from freshly recov- being nearly perfectly round and containing a vascular ered and cultured conceptuses (9–16 somite pairs) but was plexus in all but the peripheralmost region, which was less abundant in the proximal region (Figs. 6A and 6B). populated by a single layer of mesenchymal cells. Based on Double immunostaining revealed additional VCAM-1- these observations, we conclude that, during the first 24 h positive cells in the distal core of the allantois, associated

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. FIG. 5. Immunostaining for Flk-1, Flt-1, Tie-1, and Tie-2 in intact allantoises of freshly recovered and cultured conceptuses and 24-h allantoic explants. Bright-field micrographs show immunolocalized proteins (brown) in the endothelial cell lineage of the allantois. All material was lightly counterstained with hematoxylin. Freshly recovered and cultured conceptuses were at approximately 9.0 dpc and contained 14–16 somite pairs. Because allantoic explants shown here were cultured for 24 h on glass coverslips, development lagged 4–5 h behind that shown in Figs. 1A and 1B. Single and double arrows point to vascular channels. (A–C) Flk-1. Allantois from a freshly recovered conceptus (A), cultured conceptus (B), and 24-h explant (C). Note the presence of intracellular spots of Flk-1 throughout most, if not all, cells of the endothelial cell lineage (described in Downs et al., 1998). (D–F) Flt-1. Allantois from a freshly recovered conceptus (D) and 24-h explant at low (E) and high (F) magnifications showing heterogeneity of staining intensity. (G–I) Tie-1. Allantois from freshly recovered conceptus at low (G) and high (H) magnifications shows Tie-1 in trophoblast derivatives of the nascent chorionic disk. (I) 24-h explant. (J–L) Tie-2. Allantois from freshly recovered conceptus (J) and 24-h explant at low (K) and high (L) magnifications show Tie-2 staining in vessels and presumptive angioblasts. (M–P) Examples of control allantoises incubated in the absence of primary antibody (“ϪAb”, M, included in all experiments in this study) or prebinding of the primary antibody with excess control peptides (“ϩcp”) against anti-Flt-1 (N), anti-Tie-1 (O), and anti-Tie-2 (P) to demonstrate antibody specificity. Both types of controls are representative of those used in experiments to detect VCAM-1 (Figs. 6E and 6F) and VEGF (data not shown), as well as Flk-1, whose specificity was demonstrated previously (Downs et al., 1998). Abbreviations: al, allantois; am, ; ch, nascent chorionic disk; e, endothelial cell lineage; m, mesenchyme. Scale bar in (L): 100 ␮m (A, B), 110 ␮m (C, D, F, H, J, L), 370 ␮m (E, I), 184 ␮m (G), 857 ␮m (K), 200 ␮m (M, N, O, P). In Vitro Vasculogenesis in the Mouse Allantois 355

FIG. 6. Localization of VCAM-1 in intact, explanted, and suspended allantoises. (A) Freshly recovered conceptus (14–16 somite pairs) and (B) headfold-stage conceptus cultured to 9 somite pairs were immunostained with antibodies against Flk-1 (blue) and VCAM-1 (brown). Similar protein patterns were found in allantoises between 8 and 12 somite pairs in both freshly recovered and cultured conceptuses (data not shown). White arrowheads, VCAM-1-positive mesothelium; white arrows, VCAM-1-positive core allantoic cells associated with, but distinct from, the Flk-1-positive vascular plexus; black arrows, Flk-1-positive endothelial cells. (C, D) Low and high magnification of anti-VCAM-1-stained (brown color, black arrowheads) headfold-stage allantois explanted to tissue culture plastic and cultured for 24 h in high serum. Black arrow (D) points to VCAM-1-negative vascular channel. (E, F). Control headfold-stage allantoises cultured alongside those in (C, D) without anti-VCAM-1 antibody (ϪAb) (E) and with primary antibody prebound with control peptide (ϩcp) (F). (G) Explanted headfold stage allantois cultured in suspension for 24 h and doubly immunostained for Flk-1 (blue) and VCAM-1 (brown). Arrows and arrowheads as described in (A, B). The horizontal bar beneath the explant indicates the putative proximal region of the allantois, a deduction based on insertion of a hair prior to culture in related experiments. Tissue in all photographs was lightly counterstained with hematoxylin. Abbreviations: m, mesenchyme; vc, vascularizing core. Scale bar in (G): 200 ␮m (A), 214 ␮m (B), 484 ␮m (C), 420 ␮m (D, E, F), 378 ␮m (G). with, but apparently distinct from, the Flk-1 vasculature top of the explanted vascular plexus (Figs. 6C–6F and data (Figs. 6A and 6B; also see results of VEGF/VCAM-1 double not shown) but not in mesenchyme. Owing to its relatively staining, below). obscure morphological nature, further support for mesothe- Explants were examined for the presence of VCAM-1 at lium’s presence in the explants was obtained after culturing 24 and 72 h. Cells containing VCAM-1 were scattered on allantoises in suspension. Under these conditions, allan-

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 356 Downs et al.

TABLE 2 Cloning Efficiencies and Colonization Patterns of Transplanted Allantoic Cells

Average No. of Average No. of donor cells Unincorp. Ectopically Unincorp. No. of cells injecteda colonizing host Cloning Endo Solitary Meso (allantois) incorporated (conceptus) Expt chimerae (ϮSEM) allantois (ϮSEM)b efficiency (ϮSEM) (ϮSEM) (ϮSEM) (ϮSEM) (ϮSEM) (ϮSEM)b

1 10 2694 539 (Ϯ220) 20.0% 314 (Ϯ132) 182 (Ϯ67) 43 (Ϯ22) 6 (Ϯ2) 3 (Ϯ2) 389 (Ϯ135) 2 9 1616 212 (Ϯ70) 13.1% 125 (Ϯ44) 73 (Ϯ23) 13 (Ϯ5) 9 (Ϯ4) 0 341 (Ϯ54) 3 8 1616 163 (Ϯ40) 10.1% 119 (Ϯ28) 37 (Ϯ12) 7 (Ϯ2) 2 (Ϯ1) 2 (Ϯ1) 292 (Ϯ43) T 27 1975 (Ϯ359) 318 (Ϯ89) 14.4% 193 (Ϯ53) 103 (Ϯ28) 22 (Ϯ9) 6 (Ϯ2) 2 (Ϯ1) 344 (Ϯ54)

Note. In two preliminary experiments, the proportion of single, doublet, triplet, and clumped (Ͼ3) donor cells was examined in trypsinized suspensions of 24-h allantoic explants prior to transplantation. For every 100 cells injected, 55 were single cells, 12 were doublets, 13 were triplets, and 25 were clumped (range, 4–16 cells; median, 5). Of 30 host conceptuses prepared for these experiments, 2 were abnormal and discarded (Experiments 1 and 2), and 1 was found not to contain any donor cells after transplantation (Experiment 1). The cloning efficiency of allantoic explants is the ratio of the number of donor cells incorporated into host allantoises to the average number of donor cells injected. Sites of ectopic incorporation were limited to the mesothelial layer of the and amnion. Abbreviations: Endo, endothelium; Meso, mesothelium. a The number of injected cells was calculated from the average number of cells in a 24-h explant of genotype lacZ/ϩ (footnote in Table 1). The numbers of pooled allantoises for each of the three experiments were 10 (Experiment 1), 6 (Experiment 2), and 6 (Experiment 3). b The median number of donor cells colonizing the allantois was 176 and that of unincorporated donor cells outside of the allantois was 323.

toises round up and, as judged by morphology, consist of a When coexamined with Flk-1, VEGF was found, as ex- vascularizing core encapsulated by a rind of putative me- pected, in many, but not all, Flk-1-containing cells (data not sothelium (Downs et al., 1998). Double immunostaining of shown). VEGF was also found in core cells associated with, suspended allantoises with both anti-Flk-1 and anti- but distinct from, the Flk-1 vasculature. Other core cells VCAM-1 revealed a deeply VCAM-1-positive outer rind and were VEGF/Flk-1-negative. To discover whether VEGF- a short tract of VCAM-1-negative mesothelium adjacent to expressing cells also contained VCAM-1, double immuno- the proximal region, the latter of which was confirmed by staining was carried out. Both VEGF and VCAM1 were insertion of a hair into the base of the allantois prior to robustly expressed in distal mesothelium and some core culture (Fig. 6G). In addition, many core cells exclusive of cells distinct from the vasculature (Figs. 7A and 7D), the Flk-1-positive vasculature were intensely VCAM-1- consistent with the view that VEGF is not expressed in positive. Thus, localization of VCAM-1 to the mesothelium endothelial cells (Miquerol et al., 1999). This result also of ex vivo and suspended isolated allantoises suggests that confirms our view (previous section) that VCAM1- the mesothelium of explanted allantoises survives and that expressing cells are likely to be distinct from those of the scattered expression of VCAM1 on top of plated explants nascent allantoic vasculature. identifies mesothelium. Moreover, even suspended allan- Mesenchyme. The presence of mesenchymal cells in toises maintain distal-to-proximal identity. the allantois had been previously surmised only on the The mRNA of a second gene, vascular endothelial growth basis of colonization patterns following transplantation factor, had previously been identified in allantoic mesothe- (Downs and Harmann, 1997). In this study, mesenchymal lium (Dumont et al., 1995). In this study, VEGF protein was cells underlay the explants and were most conspicuous at examined in intact allantoises from both freshly recovered the perimeter of the circular allantoic mass (Figs. 1 and 5–7). and cultured conceptuses between 5 to 6 and 8 to 9 somite They appeared negative for expression of the endothelial pairs, which encompassed allantoic development during and mesothelial genes described above. Our observation and just after union with the chorion. The antibody used that single or small clusters of Flk-1-containing angioblasts recognized three murine VEGF isoforms, 120, 164, and 188. were found on top of mesenchyme at the periphery of all 24- VEGF was localized to both mesothelial and core allantoic and 72-h explants (Figs. 1B and 1D), and never alone, cells (Fig. 7A); the highest levels were found throughout suggests that association between these two cell types may both of these cell populations in the distal region (compare be required for the survival and/or maintenance of angio- Figs. 7A–7D). blasts. In 24- and 72-h high-serum explants, VEGF was scattered throughout the explant and, similar to VCAM-1, appeared Transplantation of 24-h Explants to be restricted to mesothelium where it was often, but not always, associated with morphologically distinct vessels The aforementioned findings revealed that allantoic ex- (Figs. 7E and 7F). VEGF was not found in mesenchyme plants grown in high serum mimic normal allantoic devel- (below). opment: (i) explanted allantoises vascularized with distal-

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. In Vitro Vasculogenesis in the Mouse Allantois 357 to-proximal polarity, (ii) timing of explant vascularization (Fig. 8F). Mesothelial cells were readily identifiable as was similar to that in vitro, (iii) gene expression in explants integrated cells in the epithelial sheath covering the allan- was similar to that of ex vivo and cultured intact allan- tois (Fig. 8F). Previous studies demonstrated that solitary toises, (iv) three allantoic cell lineages were represented in cells (Fig. 8F) could belong to either the mesenchymal the explants, and (v) explants proliferated. To provide sup- (Downs and Harmann, 1997) or the angioblast (Downs et porting evidence that culture in isolation did not grossly al., 1998) cell populations. alter differentiation of allantoic cells, transplantation of Thus, the majority of integrated cells were endothelial, explanted allantoises into a normal allantoic environment the fewest were found in the mesothelium, and others were was attempted. solitary, not part of the endothelium or mesothelium (Table Allantoises were removed from lacZ/ϩ transgenic con- 2). Although the number of cells belonging to each cell ceptuses, cultured on plastic for 24 h, after which all lineage is not known, selection and/or morphogenetic con- explanted cells were judged to have retained X-gal activity straint during transplantation cannot be ruled out as an (Fig. 8E). lacZ-expressing explants were then dissociated explanation for the disproportionately large numbers of and the cell suspension was transplanted into allantoises of integrated endothelial cells observed in the chimerae. Do- cultured nontransgenic host conceptuses, which had been nor cells were never found in the fetus, supporting previous cultured from the headfold stage alongside the explants. results that bulk flow of allantoic mesoderm is from proxi- The unvascularized yolk sac region of 28 normal operated mal to distal (Downs and Harmann, 1997). Outside of the conceptuses was pierced with a glass capillary (Fig. 8A), allantois, a few donor cells were found integrated ectopi- which was then directed into the host allantois (Fig. 8B); cally into the yolk sac and amnion and were limited to the donor cells were then gently blown in. Control and operated mesothelial cell layer (Table 2). conceptuses were returned to culture for an additional 8 h Small numbers of unincorporated donor cells were found to allow time for integration of donor cells, after which within the host allantois (Table 2). These donor cells 27/28 operated hosts (Fig. 8C) were judged to contain donor exhibited morphological profiles similar to those of unin- cells following X-gal staining and histological preparation corporated cells outside of the allantois and, thus, were not (see Materials and Methods). considered chimeric (compare Figs. 8G and 8H). That unin- The cloning efficiency of incorporated allantoic cells was corporated allantoic cells were not true integrants was about 15% (Table 2). Donor cell colonization was spread supported by the absence of associated red blood cells, in over the middle and distal regions of host allantoises, agreement with previous findings (Downs et al., 1998). including the fusion junction (Fig. 8D). As described previ- About 18.2% of donor cells were identified as uninte- ously (Downs and Harmann, 1997), donor cell incorporation grated clumps (Table 2) outside of the allantois, (i) free in was judged by evidence of true chimerism as well as by the exocoelomic cavity (Fig. 8G), (ii) associated with but not classification of incorporated donor cells on the basis of adherent to the yolk sac or amnion in the exocoelom, (iii) morphology and site of integration. For example, donor within the ectoplacental cone, or (iv) within the lumen of cells were scored as endothelial only if they were found the gut, possibly as a result of adherence to the outer surface together with nontransgenic cells in nascent blood vessels of the conceptus during injection and internalization as the and only if the blood vessel contained host red blood cells gut formed. Based on these calculations, we conclude that

FIG. 7. Localization of VEGF in intact and explanted allantoises. (A–D) Intact allantois of a freshly recovered 8-somite-pair conceptus doubly stained for VCAM-1 (blue) and VEGF (brown) showing the allantoic distal (d), middle (m), and proximal (b, basal) regions (A); the proximal region alone (B); the middle region (C), where the black arrows point to proximal location of VEGF in mesothelial cells; and the distal region (D), where the white arrowhead points to the apical location of VCAM-1 in mesothelium and the black arrowhead points to a small cluster of doubly positive VEGF/VCAM-1 core cells. Similar expression patterns were found in freshly recovered and cultured conceptuses between 6 and 12 somite pairs, though in cultured conceptuses, VEGF in the proximal mesothelium was of intensity similar to that in the distal region (data not shown). (E) Low and (F) high magnification of 24-h explant stained for VEGF. White arrows in (F) point to individual and clusters of VEGF-containing cells. Other abbreviations: ch, chorion; ps, primitive streak; ys, yolk sac. Scale bar in (F): 160 ␮m (A), 80 ␮m (E), 40 ␮m (B, C, D, and F). FIG. 8. Transplantation of cultured donor lacZ/ϩ allantoic cells into cultured host conceptuses. (A) 8.75-dpc cultured benzidine-stained (Downs and Harmann, 1997) host conceptus viewed from the region of the ectoplacental cone to show the vessel-free region in the yolk sac (white arrow) and selected for injection of donor cells into the allantois. (B) Schematic diagram of cultured 8.75-dpc conceptus in sagittal orientation showing the region of injection of donor cells into the host allantois. (C) Operated 8.75-dpc host conceptus cultured for a further 8 h after donor cell injection, viewed following removal of its yolk sac. (D) Schematic diagram of the fetus in (C) showing the regions of the allantois used to score the location of blue donor cells. (E) 24-h plated lacZ/ϩ explant stained with X-gal. (F) Chimeric allantois of an operated conceptus showing chimeric blood vessel with incorporated donor endothelial cells (thick arrow), host red blood cells (small arrowheads), small cluster of solitary donor cells (thin arrow), and a single donor mesothelial cell (large arrowhead). (G, H) Clumps of unintegrated donor cells (white arrows) contained within the allantois (G) and the exocoelomic cavity (H) of chimeric hosts. Host cells in (F–H) were counterstained with nuclear fast red. Abbreviations: al, allantois; b, brain; ch, chorion; h, heart; p, chorioallantoic placenta; s, somites. Scale bar in (H): 667 ␮m (A, C), 200 ␮m (E, F, H), 100 ␮m (G).

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 358 Downs et al.

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. In Vitro Vasculogenesis in the Mouse Allantois 359

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 360 Downs et al. the remaining 67.4% of injected cells were lost during small clusters of Flk-1-, Flt-1-, or VCAM-1-positive cells transplantation. (Fig. 9H). Together with aforementioned findings, the results of Thus, the inability of allantoic explants to survive be- transplantation provide further evidence that allantoic ex- yond 24 h in low serum suggests that maturing allantoises plants develop normally during the first 24 h of culture. are increasingly dependent upon external factors for main- tenance and expansion. Culture of Allantoic Explants in Reduced Serum Effect of VEGF on Low- and High-Serum Explants Early in the study, growth and differentiation of allantoic 164 explants were examined in medium containing 5% FCS As VEGF plays a major role in the developing vasculature (low serum), selected for its availability and relatively low (Carmeliet et al., 1996; Ferrara et al., 1996), recombinant cost. Because so little was known about allantoic cell VEGF164 was tested for its ability to rescue low-serum lineages, we had no a priori reason to know whether explants. An ELISA-based assay quantified the amount of reduced serum would sustain optimal explant develop- all three VEGF isoforms, 120, 164, and 188, in high-serum ment. However, preliminary experiments had revealed that medium alone, as well as in high-serum media containing explants grown in medium containing 50% FCS for 72 h single 24- and 72-h explants (Table 1). behaved similar to those grown in rat serum, though the We verified the presence of VEGF in high-serum explants former had a tendency to contract by 72 h rather than at 72 h (Fig. 10A) but discovered very few VEGF-containing remain flat on the tissue culture dish. cells in low serum (Fig. 10B). No perceptible morphological Nearly all low-serum allantoises survived the first 24 h in differences were noted between explants after 24 h in the culture, but cell numbers were lower than those of controls presence or absence of exogenous VEGF (data not shown). at the end of this period (Table 1). Intriguingly, the gross For rescue, VEGF164 (0.02, 2, 10, and 50 ng/ml) was added to morphological appearance of low-serum explants was strik- low-serum media at the start of culture. Complete replen- ingly different (compare Fig. 9A with Fig. 3E). First, mesen- ishment at 24-h intervals of culture media containing chymal cells presented a well-circumscribed, rather than exogenous VEGF resulted, by 72 h, in higher numbers of diffuse, border. Second, a vascular plexus had formed, surviving cells relative to explants cultured in 5% FCS though a central core of morphologically nondescript round alone (Table 1). Rescued explants contained VEGF (Fig. cells (Fig. 9A) largely obscured it. Immunostaining revealed 10C), Flk-1 (Fig. 10D), and Flt-1 (Fig. 10E) but, rather than that the core contained loosely attached cells, many of forming vascular channels, they formed sheets of cells with which were lost during processing. Of the cells that re- a few vascular sprouts emanating from the periphery (Figs. mained, many contained Flk-1, Flt-1, VCAM-1 (Figs. 9B– 10C–10E). Intriguingly, VCAM-1-containing cells were rare 9G), VEGF, Tie-1, and Tie-2 (data not shown), suggesting (Fig. 10F). that they were part of the endothelial and, possibly, the These findings revealed several new insights into allan- mesothelial cell lineages. Loss of the core upon immuno- toic development. First, VEGF plays a role in survival of the processing exposed the plastic underneath the explant, allantoic endothelial cell lineage. Second, the endothelial revealing that mesenchyme was not associated with them cell lineage cannot sustain allantoic mesothelium. Third, (Figs. 9B–9G). Despite feeding, low-serum explants con- mesenchyme was not able to maintain the endothelial cell tained only about half the number of cells relative to lineage in the absence of mesothelium. Last, because the high-serum controls (Table 1) and, by 72 h, consisted morphology of the rescued angioblasts was abnormal, other predominantly of attached mesenchymal cells and/or very isoforms of VEGF may be required for appropriate allantoic

FIG. 9. Culture and immunostaining of allantoic explants in low serum. Headfold-stage allantoises were cultured low-serum medium, examined for morphology (A), fixed, immunostained (brown color), and lightly counterstained with hematoxylin after 24 (B–G) or 72 h (H). The immunostained proteins are indicated in each panel. Note the reduction or absence of core explant cells in (B–G), the internal spot of Flk-1 activity in (C), the heterogeneity of staining in vascular channels in (B–E), and the absence of immunostain in mesenchyme. Black arrows indicate vascular channels (A–E), arrowheads point to VCAM-1-positive cells (F, G), the asterisk in (A) indicates the core of morphologically nondescript cells, and the white arrows (H) point to Flk-1-positive cells. Abbreviations as in Fig. 1. Scale bar (H): 290 ␮m (A), 200 ␮m (B, D, F), 820 ␮m (C, E, G, H). FIG. 10. Explanted allantoises cultured in low and high serum and in the presence of exogenous VEGF. (A–C) Bright-field micrographs of 72-h headfold-stage allantoises cultured in high serum (A), low serum (B), and low serum plus VEGF (10 ng/ml) and immunostained for VEGF (C). (D–F) Bright-field micrographs of 72-h headfold-stage allantoises cultured in 5% FCS for 72 h in the presence of VEGF (10 ng/ml) and immunostained for Flk-1 (D), Flt-1 (E), and VCAM-1 (F). (G) Phase-contrast micrograph of headfold-stage allantois cultured for 72 h in high serum in the presence of VEGF (50 ng/ml). (H) High-magnification bright-field micrograph of headfold-stage allantoic explant cultured for 72 h in high serum plus 50 ng/ml VEGF and immunostained for Flk-1. Arrows point to brown immunostained cells. Abbreviations: e, endothelial cell lineage; m, mesenchyme; ϩ, presence of VEGF (10 or 50 ng/ml); 5%, medium containing 5% FCS (low serum); 50%, medium containing 50% rat serum (high serum). Scale bar (H): 200 ␮m (A–E, G), 843 ␮m (F, H).

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. In Vitro Vasculogenesis in the Mouse Allantois 361 vascularization. Alternatively, mesothelium, which was 1997). Thus, distalmost allantoic cells are the oldest, and not rescued, may provide additional growth factors and/or proximal cells are the youngest. Differentiation of allantoic architectural cues for correct patterning of the allantoic mesoderm into endothelium and mesothelium normally vasculature. begins in the distal region and gradually spreads to the base To discover whether VEGF also plays a role in prolifera- (Downs and Harmann, 1997; Downs et al., 1998). Remark- tion of allantoic angioblasts, allantoises cultured in high ably, polarity of differentiation was maintained in allantoic serum were supplemented with VEGF (10 and 50 ng/ml). explants, the distal region being first to exhibit morphologi- VEGF at 10 ng/ml appeared to have no effect on allantoic cal vessels and the proximal region, last. Even suspended growth (Table 1) or explant morphology, which was little allantoises, which round up, maintained distal-to-proximal changed over time. Absence of reduction in vascular com- gene expression (Fig. 6G). Given their lack of connection plexity and luminal widening (data not shown) is consistent with the streak, it was therefore not surprising that little with VEGF’s role in preventing cell death. However, higher cell intermingling was found during the initial culture VEGF concentrations (50 ng/ml) resulted in excess cells period. Thus, that allantoic explants proliferated and differ- (Table 1) obscuring the vasculature by 72 h (Fig. 10G); entiated in isolation argues against a major role for the immunohistochemistry with anti-Flk-1 (Fig. 10H) and anti- primitive streak in these processes. Rather, we hypothesize Flt-1 (data not shown) confirmed the majority of these cells that, once it has entered the allantoic compartment, allan- as angioblasts. toic mesoderm may differentiate in response to its age and Taken together, these results demonstrate that the allan- toic endothelial cell lineage is responsive to VEGF and that its position. VEGF influences survival, proliferation, and morphogenesis of allantoic angioblasts in a dose-dependent manner. In contrast, VEGF cannot rescue mesothelium, whose pres- The Endothelial Cell Lineage of the Allantois ence may ultimately be required for correct morphogenesis of the allantoic vasculature. In the absence of mesothelium, The most conspicuous cell lineage in both intact and mesenchyme cannot support vascular cells. explanted allantoises was endothelial, comprising flk1 (VEGFR-2), flt1 (VEGFR-1), tie1- and tie2 (TEK)-expressing angioblasts, and vascular channels whose patterns and DISCUSSION levels of expression accorded with each other at similar developmental stages. Moreover, the presence of each of Cell proliferation, differentiation of mesoderm into an- these gene products in trophectoderm was not due to gioblasts, and angioblast coalescence and movement are vascularization of the chorion by the allantois, lending major requirements in the formation of blood vessels. The credence to the hypothesis that some trophoblast cells may cell–cell interactions essential for stimulating and coordi- function as an endothelium (Yeh and Kurman, 1989). nating these events are largely unknown for any vascular- Morphological vascularization of the explants initially izing system, least of all for the developing allantois, one of resembled a capillary net, consisting of vessels of varying three major vascular systems in the conceptus. Although thickness. Vessel sprouting was visible in the majority of most references to umbilical development in the literature 24-h explants as files of nuclei contained within long are anecdotal, a few studies have nonetheless demonstrated strands of Flk-1-, Flt-1-, Tie-1-, or Tie-2-positive cells which in the allantois some of the same gene products essential for connected one region of the net to another. Blebs of coa- development of the cardiovascular and vitelline systems lesced angioblasts were conspicuous throughout explants at (Yamaguchi et al., 1993; Shalaby et al., 1995; Sato et al., all time points examined (Downs et al., 1998; Fig. 1) and et al., 1995; Fong 1995). Thus, notwithstanding its preemi- were likely the result of vessel contraction and/or rupture nence as a fetal life-support system, the murine allantois during angioblast movement. By 72 h, the allantoic plexus may reveal global paradigms concerning the ontogeny of became considerably less complex and was accompanied by vascularity, especially given that allantoic vascularization widening of vessel lumina. How remodeling occurred is not is not accompanied by intrinsic erythropoiesis (Downs et known, but may have been the result of angioblasts merg- al., 1998). ing, with concomitant coalescence of their vacuoles. Alter- natively, vessels may have split in a process called intusus- Differentiation of Allantoic Mesoderm May Be sceptive growth, known to occur in the chick Dependent upon Cell Position and Cell Age (Patan et al., 1993). Whatever Fate mapping has revealed that the allantois grows from the mechanism of remodeling, the timing of vascular reduc- its proximal region (Tam and Beddington, 1987; Lawson et tion in vitro was remarkably similar to that in vivo when, al., 1991; Kinder et al., 1999) by addition of mesoderm from by approximately 11.0 dpc, a single umbilical artery and the primitive streak and by continuous cell proliferation vein are present (Kaufman, 1992; K. Downs, unpublished (Downs and Bertler, 2000). As new cells are added to the data). Thus, daily morphological changes in vascular mor- allantois from the primitive streak, older cells are pushed phology provided evidence that allantoic cells underwent into more distal allantoic regions (Downs and Harmann, active cell movements in isolation.

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VEGF in Survival, Proliferation, and Movement consistent with a role for VEGF in preventing apoptosis. of Allantoic Angioblasts More VEGF produced significantly greater numbers of an- gioblasts, which did not form sheets, suggesting that VEGF Two sources of VEGF had been previously documented in stimulates the passage of quiescent cells from G1 into S intact allantoises (Dumont et al., 1995; Miquerol et al., phase of the cell cycle. 1999): mesothelium, which comprises the outer sheath in VEGF may also play an additional role in cell movement intact allantoises, and cells of the allantoic core that are of angioblasts within the allantois. Extensive cell move- distinct from the vasculature. In this study, levels of VEGF ments within allantoic explants may largely be attributed protein were graded along the length of the allantois in both to binding of VEGF to its receptors, Flk-1 and Flt-1, with sites, with lowest amounts in the base and highest amounts subsequent signaling and downstream activation of allan- in the distal region during this same time period (Figs. toic cell motility (Barelon et al., 1996; Yoshida et al., 1996). 7A–7D). Variation in the amount of VEGF along the allan- Together, these data demonstrate that the allantoic en- toic axis might play a role in chemotaxis, possibly ensuring dothelial cell lineage is responsive to VEGF in a dose- bulk flow of angioblasts toward the chorion, which the dependent manner. VEGF is required for allantoic angio- allantois is thought to vascularize. Also, because previous blast survival, proliferation, and morphogenesis. Future results failed to find apoptotic cells at this time in develop- studies will document where and which VEGF isoforms are ment (Ellington, 1985; Downs and Bertler, 2000), VEGF’s present at any developmental time, in order to elucidate widespread expression in the allantois is consistent with its each one’s function in vascularization. role in stimulating angiogenesis and preventing apoptosis (reviewed in Neufeld et al., 1999). Why two allantoic cell types express VEGF is not clear. From Mesenchyme to ...? VEGF’s basal rather than apical positioning within intact Most, if not all, blood vessels begin as endothelial tubes mesothelial cells (Fig. 7) suggests almost certain secretion that, with maturity, become coated with vascular smooth into the allantoic core. However, because some isoforms of muscle cells and/or pericytes, all of which are maintained VEGF may not diffuse over long distances (Ferrara et al., in a connective tissue matrix produced by fibroblasts. The 1995), mesothelial VEGF may be limited to support of principal early function of vascular smooth muscle is to adjacent peripheral angioblasts, with deeper ones requiring secrete matrix components of the vessel wall and, later, to a more local source of growth factor. Alternatively, me- become contractile (reviewed in Owens, 1995). Pericytes sothelium and core cells may express different VEGF iso- are a heterogeneous group of cells which play an important forms or combinations of these, which differ in their role in homeostasis of blood vessels (reviewed in Nehls and isoelectric points and affinity for heparin (reviewed in Drenckhahn, 1993). Ferrara et al., 1995). Where are these supporting cells in intact allantoises and In accord with VEGF’s specificity as a growth factor of the allantoic explants? Although still disputed (Gittenberger-de endothelial cell lineage (reviewed in Neufeld et al., 1999), Groot et al., 1999), several lines of evidence point to

VEGF164 rescued allantoic angioblasts, but not mesothe- mesenchyme as the primary progenitor tissue of smooth lium, in low serum. On the basis of this result, it follows muscle cells (reviewed in Noden, 1990; Nehls and Drenck- that development of allantoic mesothelium may be inde- hahn, 1993; Owens, 1995; Gittenberger-de Groot et al., pendent of the angioblasts. Intriguingly, the rescued pheno- 1999). However, a recent study has suggested that Flk-1- type of the explanted vasculature was not normal, but containing cells may have the developmental potential to sheetlike, reminiscent of hypervascularization in the chick differentiate into smooth muscle (Yamashita et al., 2000). (Drake and Little, 1997) or disorganization of stress actin The existence of mesenchyme in intact allantoises had fibers in endothelial cells (Waltenberger et al., 1994). One previously been obtained indirectly, by noting that cells possible reason for this result is that high concentrations of from all regions of headfold-stage allantoises colonized

VEGF164 may have produced excessive signaling and mis- “periaortic mesenchyme” after ectopic transplantation into production of VE-cadherin, one of VEGF’s downstream the fetus (Downs and Harmann, 1997). In allantoic ex- targets, required for epithelialization of angioblasts (Esser et plants, the monolayer of cells beneath the vasculature was al., 1998). Preliminary experiments revealed the presence of classified as mesenchymal because (i) these cells were VE-cadherin in intact and explanted allantoises (K. Downs, present as spindle-shaped adherent cells at the periphery of unpublished data). Another possibility is that other iso- the explants, (ii) they did not express any of the genes forms of VEGF and/or other growth factors are required for analyzed in this study, (iii) they survived and proliferated in correct architectural development of the allantoic vascula- low serum, and (iv) some of them took up, incorporated, and ture. Alternatively, mesothelium is required not only as a expressed GFP via calcium phosphate precipitation (K. source of VEGF, but also to promote and/or stabilize epi- Downs and C. Czajkowski, unpublished data), typical of thelialization of allantoic angioblasts. fibroblasts. High-serum explants responded to excess VEGF in a Further study of allantoic explants may turn out to be dose-dependent manner. Intriguingly, 10 ng/ml VEGF did most instructive in elucidating the origin and function of not affect explant growth (Table 1) but did prevent reduc- endothelial-supporting cells. For example, smooth muscle tion in vascular complexity and widening of vessel lumina, has not, to our knowledge, been described in the allantois.

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If it is found, then allantoic explants may provide a most Carmeliet et al. (1996). Abnormal blood vessel development and tractable system for discovering its origin and mechanism lethality in embryos lacking a single VEGF allele. Nature 380, of formation. 435–439. Downs, K. M. (1998). The murine allantois. In “Current Topics in Developmental Biology” (R. Pedersen and G. Schatten, Eds.), Vol. CONCLUDING REMARKS 39, pp. 1–33. Academic Press, New York. Downs, K. M., and Davies, T. (1993). Staging of gastrulation in Findings presented here suggest that allantoic explants mouse embryos by morphological landmarks in the dissection are a unique and potentially powerful system for elucidat- microscope. Development 118, 1255–1266. ing many major aspects of vascular cell biology. Owing to Downs, K. M., and Gardner, R. L. (1995). An investigation into its simplicity of structure, nascent tissue homogeneity, and early placental ontogeny: Allantoic attachment to the chorion is growth as an isolated projection, the murine allantois offers selective and developmentally-regulated. Development 121, the potential to discover how the endothelium and its 407–416. supporting cell lineages differentiate and become coordi- Downs, K. M., and Harmann, C. (1997). Developmental potency of nated into a functioning embryonic organ. Explanted allan- the murine allantois. Development 124, 2769–2780. Downs, K. M., Gifford, S., Blahnik, M., and Gardner, R. L. (1998). toises may provide insight into the mechanisms by which The murine allantois undergoes vasculogenesis that is not ac- endothelial cells expand and undergo remodeling and pos- companied by erythropoiesis. Development 125, 4507–4521. sibly reveal clues concerning how arteries and veins acquire Downs, K. M., and Bertler, C. (2000). Growth in the pre-fusion their distinct identities. The ability of transplanted allan- murine allantois. Anat. Embryol. 202, 323–331. toic explants to colonize the endothelium of intact allan- Drake, C. J., and Little, C. D. (1999). VEGF and vascular fusion: toises may prove therapeutically valuable for in utero gene Implications for normal and pathological vessels. J. Histochem. therapy in cases in which a blood-borne circulating factor Cytochem. 47, 1351–1356. might be engineered to be expressed from, for example, the Dumont, D. J., Fong, G.-H., Puri, M. C. Gradwohl, G., Alitalo, K., Tie-2 promoter (Schlaeger et al., 1995) and delivered to the and Breitman, M. L. (1995). Vascularization of the embryo: A fetal bloodstream to ameliorate or cure certain fetal defects study of flk-1, tek, tie, and vascular endothelial growth factor (suggested in Downs, 1998). expression during development. Dev. Dyn. 203, 80–92. Ellington, S. K. L. (1985). A morphological study of the develop- ment of the allantois of rat embryos in vivo. J. Anat. 142, 1–11. ACKNOWLEDGMENTS Esser, S., Lamugnani, M. G., Corada, M., Dejana, E., and Risau, W. (1998). Vascular endothelial growth factor induces VE-cadherin The authors are grateful to Dr. Kaci Hocking for technical help tyrosine phosphorylation in endothelial cells. J. Cell Sci. 111, in the initial stages of this work; to Carey Bertler, Yanira O’Neill 1853–1865. Naumann, and Brendon Nacewicz for assistance with animal Ferrara, N., Heinsohn, H., Walder, C. E., Bunting, S., and Thomas, husbandry and immunohistochemistry; and to Professor Richard G. R. (1995). The regulation of blood vessel growth by vascular Gardner, Dr. N. T. Sato, and the two anonymous reviewers for endothelial growth factor. Ann. N. Y. Acad. Sci. 752, 246–256. valuable comments on the manuscript. Funds were provided (to Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O’Shea, K.M.D.) by The Graduate and Medical Schools of the University of K. S., Powell-Braxton, L., Hillan, K. J., and Moore, M. W. (1996). Wisconsin, a grant to the University of Wisconsin–Madison Medi- Heterozygous embryonic lethality induced by targeted inactiva- cal School under the Howard Hughes Medical Institute Research tion of the VEGF gene. Nature 380, 439–442. Resources Program for Medical Schools, a program project pilot Fong, G.-H., Rossant, J., Gertsenstein, M., and Breitman, M. L. grant to the University of Wisconsin from the NIEHS, the Indus- (1995). Role of the Flt-1 receptor tyrosine kinase in regulating the trial and Economic Development Research Program (University assembly of vascular endothelium. Nature 376, 66–70. Industrial Relations and the State of Wisconsin), a Parke–Davis Gardner, R. L., Lyon, M. F., Evans, E. P., and Burtenshaw, M. D. 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