Developmental Potency of the Murine Allantois

Development 124, 2769-2780 (1997) 2769 Printed in Great Britain © The Company of Biologists Limited 1997 DEV4843

Developmental potency of the murine allantois

Karen M. Downs* and Christine Harmann Department of Anatomy, 1300 University Avenue, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

The murine allantois is the future umbilical component of colonizing several derivatives of all three primary germ the placenta. The base of the allantois is also thought to layers. contain the future germ line. We have examined the fate Differences in the state of differentiation along the prox- and developmental potency of cells within the murine imodistal axis of the allantois were further borne out when allantois during gastrulation. the three allantoic regions were placed into the base of the lacZ-expressing headfold-stage allantoises (approxi- allantois of host conceptuses. Striking differences were mately 8.0 days postcoitum; dpc) were subdivided into observed in ﬁnal position along the proximodistal axis of the three proximodistal regions and transplanted into three host allantois. Most grafted cells translocated distally from sites in synchronous non-transgenic host embryos: the the base; however, basal donor allantoic cells translocated primitive streak at the level of prospective paraxial typically only as far as the host’s mid-region, whereas donor mesoderm, the primitive streak at the level of lateral plate allantoic tip cells typically returned to the tip, often colo- mesoderm, and the base of the allantois. After 23 hours nizing the chorioallantoic fusion junction. Together, our in culture, operated conceptuses were examined histolog- data reveal that the headfold-stage allantois may contain a ically for contribution of donor allantoic cells to the proximodistal gradient of differentiation, and raise intrigu- conceptus. None of the allantoic regions contributed to ing questions about how this gradient was established and paraxial mesoderm when placed into the fetus, but all the role it plays in umbilical vasculogenesis. three colonized the endothelium and adjacent mesenchyme of the dorsal aorta. The mid-region was most Key words: allantois, mouse, lateral plate mesoderm, paraxial efficient at colonizing endothelium, whereas the base was mesoderm, potency, extraembryonic mesoderm, primordial germ the only allantoic region to exhibit relative pluripotency, cells, angioblasts, vasculogenesis, erythropoiesis

INTRODUCTION mesoderm is loosely scattered, while proximal allantoic mesoderm is densely packed and continuous with the posterior Extraembryonic mesoderm is the first mesoderm formed streak. The allantois eventually meets the chorion and attaches during murine gastrulation. Epiblast cells from the proximal to it. This first step in placental morphogenesis is mediated by embryonic region of the egg cylinder translocate into the the mesothelial surfaces of the allantois and chorion nascent posterior primitive streak and emerge as a bulging (Ellington, 1985; Downs and Gardner, 1995). Fusion occurs mesodermal mass at the posterior embryonic/extraembryonic shortly thereafter and the allantois becomes overtly vascular- junction (Lawson et al., 1991). By the late streak stage (Downs ized, thereby providing the vital umbilical linkage between and Davies, 1993), this mesodermal mass develops coalescing fetus and mother. lacunae which ultimately form the exocoelomic cavity whose Because little is known about the extraembryonic mesoderm major components, the amnion, chorioallantoic placenta, and of the allantois, we initiated a study designed to address yolk sac, will support the fetus throughout the remainder of whether it is a unique mesoderm or whether it bears resem- gestation. blance to embryonic mesoderm. The possibility that cells of The allantois develops from a small bud of extraembryonic the allantois might even be pluripotent, and thus able to con- mesoderm emanating from the posterior streak at the angle tribute to other germ layers, was considered. The headfold- between the yolk sac and amnion in the early neural plate stage allantois was selected for this study, the rationale being stage (approx. 7.5 days post coitum, dpc; Downs and Davies, that two of its regions appear to have different functions: the 1993; reviewed by Downs, 1997). The allantoic bud projects tip is involved in chorioallantoic fusion (Downs and Gardner, into the exocoelomic cavity and enlarges by a combination of 1995) and the base may induce or protect presumptive pri- distal cavitation and mitosis (Ellington, 1985). During mordial germ cells (PGCs) from differentiation (McLaren, outgrowth, the allantois consists of an outer layer of mesothe- 1983, 1992). To determine the developmental potential of the lium surrounding an inner core of mesoderm. Distal allantoic allantois, the allantois of lacZ-expressing transgenic mouse 2770 K. M. Downs and C. Harmann embryos (Friedrich and Soriano, 1991; see Methods) was sub- island tissue slightly distal to the level of the site of insertion of the divided into three regions, tip, middle third, and base, at the allantois with the amnion, or (2) directly through visible yolk sac blood headfold stage (approx. 8.0 dpc, Downs and Davies, 1993). islands. Individual allantoises were then placed in 0.5 ml of culture The three allantoic regions were transplanted individually into medium (Downs and Gardner, 1995) and cultured in roller culture for each of three sites in similarly-staged conceptuses: (1) the base 24 or 40-43 hours. After culture, each allantois was stained in benzidine of the allantois (called ‘orthotopic’ and ‘heterotopic’ grafts, Bed- for 30 minutes to detect hemoglobin-expressing cells (see Histochem- ical Analysis, below) and fixed briefly in 4% paraformaldehyde for 1 dington, 1981, 1982), (2) the prospective lateral plate mesoderm hour at 4¡C before gently squashing each allantois on a microscope of the fetus (‘heterotopic’ grafts), and (3) the prospective slide for visualization and counting of benzidine-positive cells. paraxial mesoderm of the fetus (‘heterotopic’ grafts). After 23 hours of whole embryo culture, operated conceptuses were Transplantation analyzed for colonization patterns of the donor tissue in order to The basalmost portion of the allantois was taken as the site of insertion discover whether donor allantoic cells conformed to the normal of the amnion (Oz«dzenski, 1967; Fig. 2). Headfold-stage donor allan- fate of these regions, in which case they would be pluripotent, toises were removed by capillary (inner diameter 90 µm) aspiration or whether they contributed to one or a small number of cell by piercing the yolk sac just distal to the site of insertion of the types, in which case they would be restricted, or possibly even allantois with the amnion (Fig. 2). Allantoises in which basal cells determined for those lineages at the time of transplantation. were not sufficiently removed from attachment to the posterior streak after allantoic aspiration were discarded. Whole allantoises which exhibited distinct basal and distal regions were then rinsed in PBS, MATERIALS AND METHODS and placed individually in trypsin/pancreatin for 5 minutes on ice (Hogan et al., 1994), after which the tip, middle third, or base was Mouse strains excised using glass needles (Beddington, 1987; Fig. 2). Donor tissue The lacZ transgene of ROSA26 (Friedrich and Soriano, 1991) was from the embryonic regions included both epiblast and primitive made hemizygous on the C57BL/6 (BL/6) genetic background by streak but not visceral endoderm. backcrossing ROSA26 lacZ/+ hemizygotes to BL/6 for 8 generations Transplantations were carried out either as a series of orthotopic (generous gift from Dr K. Gould and Professor W. Dove, University grafts only (Table 1A), or as a series of heterotopic grafts (Table 1B) of Wisconsin-Madison Medical School). Hemizygous lacZ/+ BL/6 which also included a second set of control orthotopic grafts to the males and females were subsequently intercrossed to obtain homozy- base of the allantois. The following host sites were targeted: (1) the gous lacZ/lacZ BL/6 females. Females were judged homozygous if base of the allantois, (2) the mid-primitive streak at the level of they transmitted the transgene to all of at least 15 progeny. At n=15, prospective lateral plate mesoderm, and (3) the more anterior the probability that at least one parental female is lacZ/+ is equal to primitive streak at the level of prospective paraxial mesoderm (Fig. 0.003 (Professor Robert Wardrop, UW-Madison, Department of Sta- 3). Transplantations of the mid- and distal allantoic regions to the base tistics). Homozygous lacZ/lacZ BL/6 females were then crossed with of the allantois were not strictly orthotopic, and therefore they were male mice of the CBA strain (Jackson Laboratories). Hemizygous called ‘heterotopic’ grafts. The reason for grafting into the allantois lacZ/+ males and females (both now BL/6 × CBA) were subsequently was to control for contribution to the allantois in all experiments intercrossed to obtain a homozygous breeding strain, designated where allantoic tissue was placed into the fetus (Table 1, Experimen- ROSA26* of genetic background (BL/6 × CBA). Donor embryos for tal Set B). Every experiment included unoperated conceptuses and all experiments were obtained by mating F1 hybrid females (C57BL/6 several unoperated transgenic conceptuses in order to compare the × CBA; Jackson Laboratories) with homozygous ROSA26* lacZ/lacZ extent of development between donor and host embryos (see Mor- (C57BL/6 × CBA) males. Host embryos were non-transgenic F2s of phology of Cultured Conceptuses, below), and to ensure appropriate hybrid F1 (C57BL/6 × CBA) matings. benzidine and X-gal staining (see Histochemical Analysis). Mice were kept in 12-hour light-reversed conditions (dark period: At the time of transplantation, donor tissue was placed in a drop of 13.00-1.00 hours). Pregnant females were killed by cervical disloca- dissection medium on a glass slide from which the single chamber tion without prior anesthesia, and the uterine horns dissected into was removed, leaving behind a silicon gasket (Nunc). The tissue was phosphate-buffered saline (PBS; Sigma). then cut further into clumps of approximately 10-30 cells. Every effort Removal and dissection of conceptuses Host conceptuses were dissected at 10.00 am on the day of the experiment (approximately 7.75 days postcoitum; dpc). In most experiments, only neural plate/late allantoic bud and very early headfold-stage conceptuses were selected as hosts (Fig. 1A; Downs and Davies, 1993; Downs and Gardner, 1995) to ensure that by the time of transplantation several hours later, most embryos would be at the headfold stage. Conceptuses were paired, and the ectoplacental cone of one member of each pair was trimmed with scissor-motion of two 28 gauge syringe needles. Trimming the ectoplacental cone enabled the identification of each conceptus following transplantation and culture (Lawson et al., 1991). Pairs of host conceptuses were then placed in culture medium and maintained in the incubator (Downs and Gardner, Fig. 1. Headfold-stage donor and host conceptuses, before and after 1995) until transplantation. transplantation. (A) Headfold stage conceptus. (B) 4 µm histological section through a donor headfold-stage conceptus of ROSA26* lacZ/+ genotype stained Culture of allantoises in isolation for β-galactosidase activity. (C) Dorsal view of an operated conceptus 23 hours Allantoises of lacZ/+ hemizygous genotype were removed after whole embryo culture. Most of the yolk sac has been removed. Arrows in A- through two vitelline sites (Fig. 2): (1) through non-blood C point to the allantois. Scale Bars (A,C) 200 µm. (B) 100 µm. Developmental potency of the murine allantois 2771 AAAAAAAAAA culation, heart beat, turning, extent of development of the gut, brain, AAAAAAAAAA heart, and numbers of somite pairs (Fig. 1C). None of the conceptuses AAAAAAAAAA were grossly deficient in any one of these features, but a few exhibited 2 an obvious lack of growth and organization and were judged abnormal AAAAAAAAAA (0.65% total cultured conceptuses). The extent of development of unoperated hosts and transgenic conceptuses was also compared in AAAAtipAAAAAA order to verify that the donor and host conceptuses developed syn- YS chronously. In this analysis, a Student’s t-test revealed no significant AAAAAAAAA1 A differences between development of headfold-stage donor and host conceptuses (scoring based on Brown, 1990; n = 18 (lacZ/+), n = 38 AAAAAmid AAAAA AAAAAAAAAA (+/+); P = 0.25, equal variances assumed, CI = 95.0%). Histochemical analysis and scoring grafted cells AAAAAbaAse AAAA After culture, conceptuses and whole allantoises cultured in isolation AAAAAAAAAA were immediately stained in benzidine to identify host hemoglobin- synthesizing red blood cells: 20 µl 30% H2O2 (Sigma) was added to AAAAM AAAAAAA 1 ml benzidine stock solution (benzidine stock solution: 50 ml 0.5 M PS glacial acetic acid plus 100 mg benzidine; Sigma); 100 µl of the AAAAAAAAAA benzidine/H2O2 mixture was added to 400 µl PBS to produce a working solution. 100 µl of the working solution was added to 300 µl AAAAAAAAAA dissection medium which contained the conceptuses (Downs and AAAAAAAAAA Gardner, 1995) and this was kept at room temperature for 5-10 minutes, after which the yolk sac was pierced and the conceptuses AAAAAAAAAA stained for 10-20 minutes more. Conceptuses and allantoises were Fig. 2. Schematic diagram of the allantois and delineation of the base then rinsed twice in PBS and fixed for 2-2.5 hours at 4¡C in 4% at the level of insertion of the amnion. The site of insertion of the paraformaldehyde. All cells of ROSA26* lacZ/+ hemizygous donor amnion (AM, thick arrow) delineated the base of the allantois from the headfold-stage conceptuses were blue after staining in X-gal, both subjacent primitive streak (PS). The solid diagonal line indicates the before (Fig. 1B) and after culture (data not shown), with the exception basalmost portion of the allantois taken for this study (Oz«dzenski, of an occasional (<0.01%) benzidine-positive donor cell (see below). 1967). The allantois was subdivided into three regions for However, fixation periods of 5 hours or more compromised the transplantation (dotted lines): the base, the mid-region and the tip. The ubiquity of X-gal staining in some cell lineages, particularly yolk sac numbers 1 and 2 indicate yolk sac (YS) sites through which the endoderm, the chorion, and trophoblast giant cells overlying the ecto- allantois was removed for the experiments described in Table 2. Site 2 placental cone. No background staining for lacZ activity was found contains blood islands whereas site 1 is typically free of blood islands in non-transgenic wildtype hosts either before or after culture (not observable at the level of the light microscope. Site 1 was the principal shown). Fixed conceptuses were rinsed 3 times in PBS (20 minutes site of yolk sac puncture through which the allantois was removed for each) and stained in X-gal (Sigma) at 37¡C for at least 15 hours (Sanes all of the transplantation experiments described. The bold line et al., 1986). Postfixation was not necessary for the retention of X- surrounding the allantois and which is continuous with the yolk sac gal. Conceptuses were examined for the presence of transgenic cells and amnion represents the mesodermal lining of the exocoelom. before processing for wax histology by conventional means. Conceptuses were sectioned at 4 µm thickness (Reichert-Jung Autocut), and the sections dried at 42¡C for at least 7 hours. Slides was made to inject all fragments from the desired region; this was suc- were dewaxed by conventional means, and counterstained in Nuclear cessful most of the time. Pairs of recipient conceptuses to be injected Fast Red (Sigma; MacGregor et al., 1995) for 5 seconds to 1 minute were placed in a second drop of dissection medium. Transplantations before mounting in Permount. Nuclei of transgenic donor cells were were carried out in a Nikon Diaphot inverted microscope with bright- counted in every section and locations noted. Operated conceptuses field and DIC optics. that contained ≥5 incorporated cells were scored as chimeric. Two Leitz micromanipulation arms were used to manipulate Only red blood cells stained pale yellow to deep orange if they holding and microinjection pipettes. Holding pipettes (OD 1.00 mm, contained hemoglobin (Fig. 4A). When co-stained with X-gal, the ID 0.75 mm) were hand-pulled and heat-polished (Beddington, 1987) overwhelming majority of transgenic red blood cells contained sig- on a deFonbrune microforge to an inner diameter of 0.05 mm. nificant amounts of X-gal precipitate; some red blood cells were Microinjection pipettes were pulled on an electrode puller (Sutter speckled blue, and very infrequently (<0.01%), a hemoglobin-positive Instruments, Novato, CA), broken to an ID of 0.03 mm on the micro- cell appeared negative (Fig. 4B). In the absence of benzidine, forge, and heat-polished. Holding and microinjection pipettes were however, all transgenic red blood cells stained blue with X-gal (data mounted on microelectrode holders (World Precision Instruments) not shown). and contained either 0.1 M KCl and silicon oil (holding pipettes) or dissection medium and silicon oil (microinjection pipettes). The electrode holders were connected to Hamilton syringes of 500 µl RESULTS (holding pipettes) or 100 µl (microinjection pipettes). All experimental embryos were photographed at the time of trans- Transplantation was used to determine the developmental plantation using a video monitor attached to a TV screen (Sony) and potency of the headfold-stage allantois. Donor allantoises of printer loaded with black-and-white heat-sensitive paper (Sony) in hemizygous lacZ/+ genotype were subdivided into three order to verify retrospectively initial embryonic stage and the site of injection. regions (Fig. 3): base, middle-third and tip. Each region was transplanted into one of three sites in each host conceptus: the Morphology of cultured conceptuses base of the allantois, the primitive streak at the level of prospec- Following culture, each conceptus was scored for the following mor- tive lateral plate mesoderm (LPM) or the primitive streak at the phological features (Brown, 1990): chorioallantoic fusion, yolk sac cir- level of prospective paraxial mesoderm (PM). These 2772 K. M. Downs and C. Harmann

A B Orthotopic grafts of the region of the primitive streak that gives rise to lateral plate mesoderm (Fig. 3B,D) resulted in colonization of a wide range of tissues (Fig. 6). Contribution to tip non-mesodermal tissues likely reflects the fact that the al al mid primitive streak was not separated from epiblast (Tam and Bed- hf base dington, 1987), as it was not known whether the allantois LPM would give rise to derivatives of all three primary germ layers rather than to mesoderm alone. Therefore, in the event that it PM did, we wished to see the full colonization profile in the controls for this prospective region. Early Headfold Early Headfold ROSA26* lacZ/+ +/+ Orthotopic transplantations of the region of the conceptus giving rise to paraxial mesoderm exhibited the lowest rate of chimerism (33.3%, Table 1A) of all orthotopic grafts. However, this number was not significantly different from the overall rate of chimerism for the other two sites. Because transplantations to this site contained a similar number of grafted cells as the other two transplantation sites, the smaller number of chimeras found at the level of prospective paraxial mesoderm is likely due to a technical limitation. Overlap between the holding and injection pipettes (Fig. 3E) was apparently significant enough to result in unstable placement of donor tissue and subsequent Fig. 3. Sites of transplantation. Donor allantoises from headfold- loss of the grafted cells to the amniotic cavity or culture stage ROSA26* lacZ/+ hemizygous donor conceptuses (A) were medium (Table 1B; compare Fig. 3E with Fig. 3C,D). Never- removed and subdivided into three regions: tip, mid-region (mid), theless, we found that this staggered arrangement of microma- and base. Clumps from each allantoic region were transplanted into nipulation pipettes was superior to a symmetric one for this each of three headfold-stage host sites in a non-transgenic host (B): region (data not shown). Colonization patterns in chimeric base of the allantois (al), prospective lateral plate mesoderm (LPM), embryos included paraxial mesoderm, as previously described and prospective paraxial mesoderm (PM). C-E illustrate the actual (Tam and Beddington, 1987; Fig. 7). transplantations into (C) the base of the allantois, (D) the prospective LPM, and (E) the prospective PM. Arrows indicate the point of Developmental potency of the allantois insertion of the uppermost portion of the microinjection pipette. hf, µ Transplantation of the allantois into the base of host headfolds. Scale Bar in E for C-E 200 m. allantoises Given the array of potentially colonizable tissues at each of the embryonic sites were selected because they had been previ- three transplantation sites, a series of experiments designed to ously characterized by transplantation (Tam and Beddington, determine the developmental potential of the allantois was 1987), and therefore offered the opportunity to compare the carried out (Table 1B). Orthotopic and heterotopic transplan- developmental potential of allantoic extraembryonic tations of the allantois into the base of host allantoises were mesoderm in sites where embryonic mesoderm was forming. used to verify accuracy of graft placement by demonstrating Most hosts were at the headfold stage at the time of trans- within each experiment that allantoic cells contributed to the plantation, but a small number had reached early somite stages allantois when placed there. In addition, they provided further (Figs 5-7). After culture, each chimera was examined for the data on the fate of the basal allantoic cells, and on the state of location of donor allantoic tissue. Benzidine staining, specific differentiation of the allantoic mid-region and tip (see below). for red blood cells containing hemoglobin, facilitated identifi- When each of the allantoic regions was placed into the base cation of host endothelial cells. of the allantois of a host (Fig. 3C), grafted allantoic cells were well-incorporated in the allantois and were often distributed Orthotopic control transplantations amongst two of the three allantoic regions delineated in this To obtain a profile of the potentially colonizable tissues for study: base, mid-region and distal third (Fig. 5). Grafted each of the three transplantation sites, a set of five orthotopic allantoic cells were typically well-spread across the width of experiments was carried out, using similar numbers of con- the host allantois in sagittal sections (data not shown). Several ceptuses (Table 1A). The number of chimeras obtained after representative examples of donor allantoic cell distribution in grafts at the orthotopic embryonic sites was within the range host conceptuses are shown in Fig. 8A,B. of that previously reported (5-12 chimeras; Tam and Bedding- Of potential significance was our finding that when placed ton, 1987). Embryos receiving grafts from the base of the into the base of the allantois, donor allantoic cells from all three allantois exhibited the highest rate of chimerism, whereas those allantoic regions were conspicuously absent in the fetus, either receiving grafts at the level of paraxial mesoderm were least as integral tissue components or contained within the fetal vas- efficiently colonized. culature. Rather, most donor allantoic grafts contributed cells Orthotopic transplantations of the base of the allantois were only to the allantois or, in a minority of cases, to the yolk sac carried out to reveal the fate of cells in the base of the allantois. (Fig. 5; see below). Moreover, only 8.2% of all donor grafts All orthotopically placed cells from the base of the allantois contributed cells to the base of the allantois. This observation contributed exclusively to the allantois. None were found in the suggests that normal cell movement within the allantois is from fetus (Fig. 5A). proximal-to-distal. Of the chimeras which contained a few Developmental potency of the murine allantois 2773

Table 1. Frequency of experimental chimeras and mean number of grafted donor descendant cells Mean no. grafted cells Type of Expt Site of Transplanted No. embs. No. neg. No. grafts No. chimeras ±s.e.m. tissue set injection tissue No. expts injected grafts (%) unincorp. (%) (%) (median) colonized A O-Base Base of allantois 5 20 3 3 14 182.6±46.2 Allantois (15.0) (15.0) (70.0) (115.5) O-LPM Prospective LPM 5 19 4 5 10 192.5±68.7 Multiple (21.1) (26.3) (52.6) (100.5) O-PM Prospective PM 5 21 6 8 7 145.9±61.3 Multiple (28.6) (38.1) (33.3) (73) B Base of allantois Base of allantois 4 15 5* 3 7 442.0±304.0 Allantois (33.3) (20.0) (46.7) (185.0) Middle third 4 21 2† 3 16 607.0±165.0 Allantois (9.5) (14.3) (76.2) (471.0) Tip of allantois 4 14 1 1 12 208.4±82.6 Allantois (7.1) (7.1) (85.7) (102) Prospect. LPM Base of allantois 4 17 7 6 4 697.0±402.0 Multiple (41.2) (35.3) (23.5) (452.0) Middle third 4 22 3 7 12 230.8±60.0 Endothelia, (13.6) (31.8) (54.5) (175) mesenchyme Tip of allantois 4 18 2 11 5 199.0±139.0 Endothelia, (11.1) (61.1) (27.8) (87) mesenchyme Prospect. PM Base of allantois 4 19 7 10 2 68.0±48.0 Endothelia, (36.8) (52.6) (10.5) mesenchyme Middle third 4 21 7 8 6 176.7±75.7 Endothelia, (33.3) (38.1) (28.6) (130) mesenchyme Tip of allantois 3 13 1 8 4 85.0±43.2 Endothelia, (7.7) (61.5) (30.8) (62) mesenchyme

In Experimental Set A, orthotopic (O) transplantations were carried out to three sites: the base of the allantois, the prospective lateral plate mesoderm (LPM), and the prospective paraxial mesoderm (PM). In Experimental Set B, the allantois was subdivided into three regions, tip, middle third, and base. Each of these regions was then transplanted to three sites in the same experiment: the base of the allantois, the prospective LPM, and the prospective PM. Abbreviations: embs, embryos; expt., experiment; neg., negative; no., number; s.e.m., standard error of the mean. *One graft had only four cells incorporated into the allantoic endothelium and was scored as negative. †One graft had only one cell incorporated into the allantoic endothelium and was scored as negative. donor cells in the base, colonization by these donor cells was contributions to the chorioallantoic fusion junction of the host found in the nascent allantoic vasculature (data not shown). (Fig. 9), with cells from the tip contributing the highest In four chimeras, some donor allantoic cells contributed to number. In contrast, donor basal cells rarely translocated that the endothelium of the immediately adjacent omphalomesen- far, typically ending up in the host allantoic mid-region where teric artery (Fig. 5A,B). Because the donor allantois was they contributed predominantly to the nascent endothelial always at the headfold stage, it is unlikely that yolk sac cells network (Figs 5A,D, 9). had infiltrated the allantois at this early time and returned to In three chimeras, nine benzidine-positive cells of donor the yolk sac after grafting (M. Blahnik, S. Gifford, and K. origin were identified amongst the benzidine-positive cells of Downs, unpublished data). Rather, because the overwhelming the allantois (Figs 4C, 5A,B). This was tantalizing, because it majority of grafts contributed exclusively to the allantois suggested that the allantois, like the yolk sac, may be intrinsi- (45/49), we believe that contribution to the yolk sac vascula- cally both erythropoietic and vasculogenic. On the other hand, ture may have been due to contamination of the yolk sac by it could mean that a few donor allantoic cells had been conta- grafted cells when the injection pipette was withdrawn from minated by yolk sac blood islands or, less likely, induced to the conceptus, or possibly by displacement of part of the graft form red blood cells during removal through the erythropoietic to the yolk sac during subsequent culture. This interpretation yolk sac. is based on one instance where a small cluster of unincorpo- To distinguish between these possibilities, allantoises of rated cells (chimera number 3, Fig. 5B) was found adherent to lacZ/+ genotype were cultured in isolation for 24 and 40-43 the outside of the yolk sac at the level of the site of injection hours (Table 2). Allantoises were removed through the yolk into the base of the allantois. sac, either just above the level of the base of the allantois (Site Striking differences were found in the pattern of col- 1, Fig. 2), as had been done for all the grafts reported here, or onization of the host allantois by each of the three donor directly through more distally situated yolk sac blood islands allantoic regions (compare Figs 5A-C; summarized in Fig. 5D). (Site 2, Fig. 2). In particular, although all three allantoic regions had been In isolation, allantoises lost their rod-like shape and were placed into the base of the allantois (Fig. 3C), cells derived round, suggesting that the unique projectile-like morphology from the donor mid-region and distal third made significant of the allantois is a result of molding of the allantois by its 2774 K. M. Downs and C. Harmann

Fig. 4. Benzidine staining in donor allantoises and location of yolk sac blood islands. (A) Double benzidine (and X-gal) staining of the yolk-sac of a non-transgenic host conceptus cultured for 23 hours. There is no background X-gal staining in the host. Arrow points to an example of a benzidine-positive red blood cell which, in this study, were brown. (B) Double benzidine and X-gal staining of transgenic donor conceptuses cultured for 23 hours. There is one red blood cell (arrow) in this area that appeared negative for staining with X-gal. (C) Two presumptive donor-derived benzidine-positive cells in the mid-allantoic region of a host conceptus cultured for 23 hours (arrows). These presumptive red blood cells were derived from grafted allantoic tissue originating in the allantoic mid- region of chimera 1, Fig. 5B. (D) Allantois removed from site 1 (Fig. 2), cultured in isolation for 43 hours, and stained in benzidine. Although this is a transgenic explant, it was not co-stained in X-gal. Note the benzidine-positive cells in vascular networks in the isolated allantois. Vascularization in allantoises cultured in suspension was, however, rare (1.0%). (E,F) Two ROSA26* lacZ/+ headfold-stage conceptuses showing variation in the distance between the yolk sac blood islands and the site of removal of the allantois (al) for grafting. The upper arrow indicates a yolk sac blood island and the lower arrow points to the level of yolk sac puncture for removal of the allantois. Note that the distance between the arrows in E is greater than the 90 µm diameter of the capillary used to remove the donor allantois, whilst in F that distance is less than 90 µm. Thus, an allantois removed from a conceptus similar to that in F would have exited the yolk sac through broken blood islands. Scale Bar in C, for A-D: 25 µm (A); 20 µm (B); 21 µm (C); 35 µm (D). Scale Bar in F for E and F 100 µm. environment during growth in the exocoelomic cavity and not could not be ruled out. The question of whether the allantois to some intrinsic polarizing activity within the allantois itself. is intrinsically erythropoietic awaits further analysis. Benzidine staining revealed the presence of hemoglobin- producing cells in allantoises removed through both yolk sac Heterotopic transplantations of donor allantoic sites (Table 2; Fig. 4D). Although there appeared to be fewer tissue positive allantoises amongst those that had been removed Prospective lateral plate mesoderm (LPM) through Site 1 (Fig. 2), the Student’s t-test revealed that this To discover whether allantoic mesoderm would become incor- difference was not signiﬁcant (P=0.069, equal variances porated into one or more of the control tissues formed at the assumed, conﬁdence interval, 95.0%, df=93). level of the primitive streak that gives rise to LPM, the tip, This comparison suggests that the allantois may be intrinsi- middle third, and base of donor allantoises were transplanted cally erythropoietic. However, from histological examination into the prospective lateral plate mesoderm of host embryos of a series of twelve headfold-stage conceptuses, it was (‘heterotopic’ transplantations; Fig. 3D). In contrast with the concluded that the distance between the yolk sac blood islands transplantations into the allantois, where most grafted cells and the site of removal of the allantois was, in many cases, less contributed to the allantois, all three allantoic regions con- than the diameter of the pipette used to remove donor allan- tributed descendants to the fetus when placed into prospective toises (Fig. 4E,F). Therefore, the allantoic graft may have been LPM (Fig. 6). contaminated with yolk sac-derived cells; alternatively, that a All three regions of the donor allantoises integrated into the small number of donor allantoic cells may have been induced endothelium of the dorsal aorta in all chimeras (Figs 6, 8C,D). to become erythropoietic during removal through the yolk sac Most grafts also colonized the mesenchyme adjacent to the

Table 2. Benzidine staining of allantoises cultured in isolation Site of removal Culture time No. positive Mean no. benzidine+ Median no. (YS) (hours) No. expts. No. explants explants (%) cells±s.e.m. benzidine+ cells 1. Non-blood islands 24 3 23 4 (17.4%) 5.8±2.9 3.5 40-43 3 35 4 (11.4%) 43.0±25.4 25.5 2. Blood islands 24 3 22 6 (27.3%) 31.5±6.4 18.5 40-43 2 18 6 (33.3%) 24.5±11.1 18.0

Donor allantoises (lacZ/+) were removed through two vitelline sites in the same experiments (shown in Fig. 2 as sites 1 and 2, and described in Materials and Methods). Isolated allantoises were then cultured for 24 or 40-43 hours, stained in benzidine and ﬁxed brieﬂy before examination in the compound microscope. Abbreviations: YS, yolk sac; s.e.m., standard error of the mean. Developmental potency of the murine allantois 2775

A Orthotopic Transplantations: Base of the Allantois (Headfold Stage)

Chimera No→ Tissue 123456789101112131415161718192021 Colonized ↓ Allantois Base + Mid Tip

Yolk Sac Omph Art Meso

No. Donor- Derived RBCs 16 in Allantois Initial Stage/Final HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ 4/ HF/ HF/ 4/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ Somite Pairs 14 13 11 10 12 11 14 11 12 12 13 16 13 12 12 15 13 15 14 12 14 of Host Embryo Experimental BBAAAAABAAABAAAAABBBA Set (Table 1) Total Number 311 Incorporated 60 2253 294 114 49 52 640 251 7 180 146 267 392 97 46 112 7 185 72 117 (54, (278, Donor Cells al) al) B Heterotopic Transplantations: Allantoic Mid-Portion to Allantoic Base (Headfold Stage)

Chimera No→ Tissue 12345678910111213141516 Colonized ↓ Allantois Base

Mid + Tip

Yolk Sac Omph Art Meso.

No. Donor- Derived RBCs 2 in Allantois Unincorp. x x (amn (ext. cav) of ys) Initial Stage/Final HF/ 5/ HF/ HF/ HF/ HF/ HF/ 4/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ Somite Pairs 12 17 9 10 8 9 8 16 12 8 14 12 5 11 12 9 of Host Embryo Total Number 70 Incorporated 1599 248 (24, 69 539 645 693 403 154 2504 628 774 32 1081 145 131 Donor Cells al)

C D Summary of Orthotopic and Heterotopic Grafts to the Heterotopic Transplantations: Base of the Allantois Allantoic Tip to Allantoic Base (Headfold Stage) Region Grafted→ Allantoic Region Base Mid-Portion Tip ↓ Chimera No→ Colonized Tissue 123456789101112 Base ++0 Colonized ↓ Allantois Base Mid-Region

Mid Distal Third

Tip Vitelline ++ Omphalomes Artery Initial Stage/Final HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ HF/ 3/ No. Chimeras with 120 Somite Pairs NS NS 11 14 NS NS 13 11 12 11 12 16 Unincorporated of Host Donor Cells Embryo Initial Stage of Host Headfold Headfold, 3-5 Headfold, 3 Total Number Embryos Incorporated 544 92 35 34 990 84 190 7 13 199 112 201 Final Number 10-16 8-16 11-16 Donor Cells Somite Pairs

Total Number of 5652 9715 2501 Incorporated 100% Transgenic Cells 75.0 - 99.9% Number Chimeras 21 16 12 50.0 - 74.9% (% Total Injected) (60.0%) (76.2%) (85.7%) 10.0 - 49.9% 1.0 - 9.9% + <1.0% Fig. 5. Grafts of donor allantoic tissue into the base of host allantoises, headfold stage. Results of transplants of the three donor allantoic regions, tip, middle third, and base, into the base of the allantois of host conceptuses are shown for every chimera scored after 23 hours in culture. For scoring, the host allantois was subdivided into the base, mid-region and tip. (A) Distribution of grafted basal allantoic cells. This ﬁgure combines the results of Table 1, in which the base of the allantois was transplanted orthotopically in two experimental sets, designated A and B. (B) Distribution of grafted donor allantoic cells whose origin was the mid-region. (C) Distribution of grafted donor allantoic cells whose origin was the distal third. (D) Summary of grafts of donor allantoic orthotopic and donor allantoic heterotopic transplantations into the base of the host allantois. HF, headfold stage; Omph Art, omphalomesenteric artery; Meso, mesoderm of the yolk sac; amn cav, amniotic cavity; ext. of ys, exterior of yolk sac. Ordinal numbers are the initial and ﬁnal numbers of somite pairs. NS, not scored. 2776 K. M. Downs and C. Harmann a 4 4 11 HF/ himer t the 4 xx y c 2 13 HF/ er eak a v p i 7 345 8 10 T HF/ or e e str v 9 wn f 8 16 HF/ imiti 12 e sho 11 751 HF/ 8 2 4/ 1 1 14 s of somites. 6 1 xx 4/ 2 1 13 ions into the pr enic cells ar g e e pair 0 4/ ansg 1 16 549 s ar 3 3/ 6 15 umber Mesoderm ee allantoic r 3/ 15 367 Allantois:

dinal n

tion of donor tr 7 Plate 2/ 8 15 tion or old; or 7 9 10 HF/ Mid-P Lateral afting of the thr r x 56789 Heterotopic: 14 , headf 253 HF/ umber and loca 10 HF/ 479 otopic g wn. HF he n ospective e) g Pr xx . T 12 HF/ 286 e e sho g of 4 1 10 vel HF/ old Sta Le m and heter old sta ptus ar 12 530 HF/ the (Headf at + 41234 10 ic conce HF/ 1826 m, headf te mesoder 12 711 HF/ Streak himer al pla Base h c 0 6 11 HF/ ter te mesoder e la 123 v 11 Primitive HF/ 192 al pla es of eac g ter the 6 0 3/ 3 1 15 ospecti m e la v into nal sta 9 2/ 15 565 ion of pr ospecti Mesoder Grafts 13 g HF/ 127 e 5 12 Plate HF/ el of pr v al he initial and ﬁ 11 HF/ 116 m. T Later t the le afting of the r e 5 thotopic: r 8 13 HF/ Or eak a 9 45678 13 HF/ e str te mesoder v Prospectiv 11 498 HF/ of al pla imiti ter 10 HF/ 432 <1.0% 100% 75.0 - 99.9% 50.0 - 74.9% 10.0 - 49.9% 1.0 - 9.9% e la Region v 2 thotopic (same site) g 123 5 14 HF/ + t , . , e p ta . ↓ m m m m a p ies ta ube ym ospecti afts into the pr ed Host T Plate Aor → ace Incor , otal s after or Aor al Gr Future T

al mediate ansgenic No essels r Tissue unction Am/All Surf Coelom Somites T Chimer V Umbilical of J Cells Ectoder Unincor Capillar Initial/Final Endoder Mesoder Mesoder Adj. Coloniz Stage Neur Dorsal . 6. Later Inter Endothelium, Intersegmen Endothelium Mesench el of pr Endothelium v ig F 23 hour le Developmental potency of the murine allantois 2777

Grafts into the Primitive Streak at the Level of Prospective Paraxial Mesoderm (Headfold Stage)

Orthotopic: Heterotopic: Allantois: Region of Prospective Paraxial Base Mid-Portion Tip Mesoderm Chimera No→ Tissue 1234567121234561234 Colonized ↓ Somites

Pre-Somitic Mesoderm Capillaries

Neural Tube

Notochordal Plate Surface Ectoderm Endothelium of Intersegmental Vessels Endothelium, Dorsal Aorta Mesenchyme Adj Dorsal Aorta Unincorp. x xxxx

Initial/Final HF/ 4/ HF/ HF/ HF/ HF/ 3/ HF/ HF/ HF/ 1/ HF/ HF/ HF/ 1/ HF/ HF/ HF/ HF/ Stage Host 12 14 13 10 13 10 14 11 9 13 13 10 13 8 12 11 13 12 12 Total Transgenic Cells 364 133 387 73 31 22 11 116 20 468 224 296 36 28 8 202 14 26 98 Incorp.

100% 75.0 - 99.9% 50.0 - 74.9% 10.0 - 49.9% 1.0 - 9.9% Fig. 7. Grafts into the primitive streak at the level of prospective paraxial mesoderm, headfold stage. This figure summarizes the number and location of donor transgenic cells 23 hours after orthotopic (same site) grafting of the region of prospective paraxial mesoderm and heterotopic grafts of the three allantoic regions into the primitive streak at the level of prospective paraxial mesoderm. The initial and final stages are as described in Fig. 6. dorsal aorta and a few exhibited donor descendants in the During the headfold stage, the primitive streak has fully endothelium of the nearby intersegmental vessels. The mid- elongated from its posteriormost site at the region of the allantois exhibited the highest rate of fetal embryonic/extraembryonic junction of the conceptus to the chimerism whereas the base alone exhibited a wider col- distal tip of the egg cylinder; it is still an active source of meso- onization range than the other two allantoic regions, with basal dermal tissue at this stage (Tam and Beddington, 1987). The cells found also in lateral plate mesoderm, surface ectoderm, developmental potency of the headfold-stage allantois was and in endoderm of the future coelom (Figs 6, 8D). challenged by placing it into two sites of the primitive streak of synchronous embryos: (1) the primitive streak where Prospective paraxial mesoderm (PM) mesoderm emerges as lateral plate mesoderm, and (2) the None of the three allantoic regions colonized presomitic or primitive streak where paraxial mesoderm is formed, just paraxial (somitic) mesoderm (Table 1B; Figs 3E, 8E) but all posterior to the node (Tam and Beddington, 1987). In addition, three again colonized the endothelial lining of the dorsal aorta placement of the distal two-thirds of the allantois into the base, and adjacent mesenchyme (Fig. 7). The allantoic mid-region originally intended to control for contribution of donor produced the highest average number of grafted cells, whereas allantoic cells to the allantois in the second set of experiments the base and tip contributed relatively few. Interestingly, the (Table 1B), provided additional data on the state of differen- base of the allantois did not exhibit the relative pluripotency in tiation of the headfold-stage allantois. this region of the conceptus that it did when transplanted into Oz« dzenski (1967) hypothesized that a small population of prospective LPM. alkaline phosphatase-positive cells that resides in the base of the headfold-stage allantois contributes either to the mature allantois, to the germ line or to both. It appears from our results DISCUSSION that the basal portion of the allantois contributes only to the mature allantois. However, these results are complicated by the We have investigated the developmental potential of the fact that there is no precise morphological feature that clearly extraembryonic mesoderm of the headfold stage allantois. defines where the base of the allantois begins. This was the 2778 K. M. Downs and C. Harmann reason that Copp et al. (1986) included the entire allantoic bud This suggests that the environment at this level of the streak in orthotopic transplantations designed to discover the where- cannot support pluripotent basal cells and more specialized tip abouts of the primordial germ cells. Although we defined the cells. It further suggests that those grafted cells that have base of the allantois according to Oz« dzenski (Fig. 2), one of become angioblasts may have the best chance of becoming us had previously shown that allantoic tissue can be regener- incorporated into this less ‘allantoic-favorable’ region of the ated following aspiration of the headfold-stage allantois conceptus. (Downs and Gardner, 1995). This argues that removal of the Grafts of allantoic tissue into the base of the allantois allantois via aspiration is either incomplete or, if it is complete, confirmed that there may be significant differences in the state cells beneath the allantois retain the potential to regenerate an allantois (Downs and Gardner, 1995). Therefore, any future studies designed to analyze fate in the base of the allantois will need to define precisely the allantoic/posterior primitive streak region. The allantois and embryonic mesoderm All three regions of the donor allantois contributed predominantly to the endothelium of the dorsal aorta and adjacent mesenchyme when placed into the fetus. This mesenchyme is thought to be recruited into connective tissue and/or smooth muscle making up the surrounding tunicae of the mature aorta (Arey, 1965; reviewed by Noden, 1989). Only the base of the allantois exhibited a greater developmental potential than the other two regions when placed into prospective lateral plate mesoderm, contributing descendants to the endoderm of the future coelom as well as to surface ectoderm and lateral plate mesoderm, similar to the orthotopic control grafts. Thus, the base of the allantois may contain a somewhat more developmentally labile population of cells than the two distal allantoic regions. The relative pluripotency of the base of the allantois is not surprising, given that cells in the base of the allantois are the most recent mesodermal cells to emerge from the posterior streak until the 3-somite stage (Tam and Beddington, 1987), and therefore, they may be the least differentiated. The fetal lineages colonized by Fig. 8. Colonization of donor tissue in host conceptuses. (A) Orthotopic transplantation of allantoic tissue following placement into the base of the allantois (chimera no. 13, Fig. 5A). Colonization of grafted descendant cells the primitive streak at the level of to the endothelium is well-distributed (arrows) through the mid- and distal region of the host prospective paraxial mesoderm revealed allantois (al). ch, chorion. (B) Heterotopic transplantation of the mid-region of a donor no overlap with the orthotopic control allantois to the base of a host allantois (al) (chimera no. 1, Fig. 5B). Arrows indicate the grafts. In particular, none of the regions of spread of the donor cells in this section. The donor cells indicated by the uppermost arrow the allantois contributed to paraxial are examples of cells scored as mesenchyme, since they did not contribute to an obvious mesoderm. This suggests that the allantois vessel in adjacent sections. (C) Heterotopic transplantation of a donor allantoic mid-region shares few, if any, properties with pre- to the primitive streak at the level of prospective lateral plate mesoderm of a host (chimera somitic, or paraxial, mesoderm. The base no. 4, Fig. 6). The contribution of grafted cells is to the endothelium (arrows) of the host aorta. (D) Heterotopic transplantation of the base of a donor allantois to the primitive streak and the tip of the donor allantois exhibited of prospective lateral plate mesoderm (chimera no. 2, Fig. 6). Contribution of donor cells is the fewest number of grafted cells per to endoderm (e), the mesenchyme (m) adjacent to the endothelium of the aorta, and to the embryo after placement into prospective endothelium of the aorta (a). (E) Orthotopic transplantation of prospective paraxial paraxial mesoderm whereas the mid- mesoderm (chimera no. 1, Fig. 7) showing contribution of grafted donor cells to host region produced the most descendants. somites (s). Scale Bar in E: 40 µm (A); 20 µm (B-E). Developmental potency of the murine allantois 2779

100 vascular systems are in place. Angiogenesis occurs during both 95 embryogenesis and throughout the life of the organism. 90 Very little is known about how the umbilical vasculature is 85 formed. Electron micrographic analyses of the murine allantois 80 have not been presented in enough detail to confirm the existence 75 Base 70 of angiogenic clusters (Tamarin and Boyde, 1976), but Ellington Mid-Portion 65 (1985) has described rudiments of the vascular system in the rat 60 Tip by 10 dpc (equivalent to approximately 4-somite pairs in the 55 mouse). Expression studies have also shown that Vascular Endo- 50 thelial Growth Factor (VEGF), its two receptors, VEGFR1 flt-1 45 and VEGFR2 flk-1 (Yamaguchi et al., 1993; Breier et al., 1995) 40 35 and TEK (tie-2) (Schnurch and Risau, 1993) are expressed early 30 in the allantois. These suggested that vasculogenesis was 25 occurring in the prefusion allantois, but without examination of % Contribution of Donor Allantoic Cells 20 the developmental fate and potency of allantoic cells, the true 15 significance of these expression patterns could not be verified. 10 Our transplantation experiments support the possibility that 5 0 the umbilical vasculature is formed by vasculogenesis rather endo1 mes2 mch3 fus4 rbcs5 than by angiogenesis. This is because allantoic angioblasts were found incorporated in the large vessel endothelium of the Host Allantoic Cell Types Colonized by Grafted Donor Allantoic Cells dorsal aorta and intersegmental arteries rather than in small Fig. 9. Colonization patterns by cell type of grafted allantoic tissue capillaries. Although it is possible that the nearby yolk sac con- into host allantoises. This histogram depicts the colonization patterns tributes endothelial cells to the allantois, making angiogenesis of donor allantoic tissue 23 hours after placement into the base of the the principal mechanism of formation of the umbilical vascu- allantois of host conceptuses. Bars represent the percentage of donor cells from each allantoic region contributing to endothelium (endo), lature rather than de novo vasculogenesis, we have some mesothelium (mes), mesenchyme (mch), the chorioallantoic fusion evidence that this is not the case. Yolk sac transplantations junction (fus), and red blood cells (rbcs). No effort was made to demonstrated that the earliest time at which yolk sac cells move distinguish cell types at the chorioallantoic fusion junction. The total into the allantois is after fusion, at about 10-somite pairs (M. number of colonizing grafted cells for each donor allantoic region Blahnik, S. Gifford, and K. Downs, unpublished data). This is was 5613 (base), 9538 (mid-portion), and 2501 (tip). approximately 22 hours after the headfold stage, the time at which the donor allantoises used in this study were removed for transplantation. Also, benzidine staining and cell profiles of differentiation along the proximodistal axis of the headfold- demonstrated that all of the yolk sac-derived cells within the stage allantois (Fig. 5D). The majority of basal allantoic cells host allantois appear to be red blood cells. moved only as far as the allantoic mid-region. In contrast, the If angioblasts are formed de novo within the allantois, then majority of donor cells whose origin was the allantoic tip how do the extraembryonic mesodermal cells that constitute moved further distally. The tip exhibited the largest fraction of the allantoic bud differentiate into angioblasts? One possibil- cells in the chorioallantoic fusion junction (Fig. 9). This ity is that distance of pluripotent mesodermal cells from the suggests that allantoic tip cells might be chorioadhesive as posterior streak dictates angioblast differentiation. Extraem- early as the headfold stage, although the full repertory of gene bryonic mesoderm destined for the allantois emerges from the expression required for chorioallantoic fusion is probably not primitive streak in a relatively pluripotent state, but as it is yet in place, as previous studies have shown that the distal half pushed distally by sustained addition of nascent extraembry- of the allantois is not competent to fuse with the chorion until onic mesoderm into the allantoic base, it moves out of the it has attained developmental maturity, typically at the equiv- sphere of influence of the primitive streak and differentiates alent of 4-6 somite pairs (Downs and Gardner, 1995). Alter- into angioblasts. Movement farther into the tip entails further natively, cells destined for the umbilical endothelial cell differentiation, either into chorioadhesive cells or possibly into lineage at the fusion junction may be further differentiated and specialized umbilical endothelial cells. possess properties different from those in the mid-region, Alternatively, differentiation of allantoic cells may be tem- which may explain why tip cells did not avidly colonize the porally, rather than spatially regulated. The fate of allantoic fetal vasculature. cells may be dependent upon the amount of time they reside in different allantoic levels, the length of which may in turn be Umbilical vasculogenesis dependent upon the rate at which cells are recruited into the Previous studies have suggested that all mesoderm has the base of the allantois and/or the number of cell cycles completed ability to undergo vasculogenesis, given the proper environ- by allantoic cells before translocating distally. ment (Pardanaud et al., 1987; Coffin and Poole, 1988). The heart, large blood vessels and the vitelline vasculature are Conclusions formed by vasculogenesis, a uniquely embryonic process in The main function of the allantois is to fuse with the chorion which pluripotent mesodermal cells differentiate into and vascularize, thereby forming the umbilical component of angioblasts that subsequently aggregate and assemble in situ the chorioallantoic placenta. Allantoic vascularization is not into new blood vessels. Angiogenesis, which is the formation dependent upon fusion with the chorion (Yamaguchi et al., of blood vessels from pre-existing ones, occurs once the major 1993; Downs and Gardner, 1995). We have found that 2780 K. M. Downs and C. Harmann angioblasts are formed almost as soon as the allantois emerges Coffin, J. D. and Poole, T. J. (1988). Embryonic vascular development: from the posterior primitive streak. Moreover, formation of the immunohistochemical identification of the origin and subsequent umbilical vasculature may not be dependent upon interaction morphogenesis of the major vessel primordia in quail embryos. Development 102, 735-748. with endoderm (Risau and Flamme, 1995) because, as far as is Copp, A. J., Roberts, H. M. and Polani, P. E. (1986). Chimaerism of known, the murine allantois consists entirely of mesoderm. Our primordial germ cells in the early postimplanation mouse embryo following discovery of the whereabouts of pluripotent and differentiated microsurgical grafting of posterior primitive streak cells in vitro. J. Embryol. cells within the allantois provides a developmental blueprint exp. Morph. 95, 95-115. Downs, K. M. (1997). The murine allantois. In Current Topics in essential for understanding the role of genes expressed in the Developmental Biology (eds. R. Pedersen and G. Schatten). New York: allantois during the headfold stage. The allantois, heretofore Academic Press. (In press). little studied, offers a promising and highly manipulable Downs, K. M. and Davies, T. (1993). Staging of gastrulation in mouse system to discover how angioblasts are formed from pluripo- embryos by morphological landmarks in the dissection microscope. tent mesoderm in the living mammalian conceptus. Development 118, 1255-1266. Downs, K. M. and Gardner, R. L. (1995). An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and This study would not have been possible without the generous developmentally-regulated. Development 121, 407-416. support and tutelage of transplantation techniques from Dr Rosa Bed- Ellington, S. K. L. (1985). A morphological study of the development of the dington. K. M. D. is also indebted to Professor Robert Auerbach for allantois of rat embryos in vivo. J. Anat. 142, 1-11. his gift of Leitz micromanipulation arms, an electrode puller, and the Friedrich, G. and Soriano, P. (1991). Promoter traps in embryonic stem cells: deFonbrune microforge. The authors are very grateful to Dr Karen a genetic screen to identify and mutate developmental genes in mice. Genes Gould and Professor William Dove for the gift of backcrossed Dev. 5, 1513-1523. ROSA26 hemizygous mice; to Melanie Blahnik for expert animal Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994). husbandry; to Shannon Gifford for Fig. 2 and for her data on allantoic Manipulating the Mouse Embryo. A Laboratory Manual. 2nd edition. Cold Spring Harbor Press: Cold Spring Harbor, New York. erythropoiesis; to Shannon Gifford and Dr James Bieker for help with Lawson, K. A., Meneses, J., and Pedersen, R. A. (1991). Clonal analysis of benzidine staining; and to Professor Matthew Kaufman for a valuable epiblast fate during germ layer formation in the mouse embryo. Development discussion on the appellation of mesenchyme adjacent to the fetal 113, 891-911. aorta. K. M. D. is especially grateful to Professor Richard Gardner for MacGregor, G., Zambrowicz, B. P., and Soriano, P. (1995). Tissue non- thoughtful and valuable comments on the manuscript and always for specific alkaline phosphatase is expressed in both embryonic and his generosity of time and discussion, and to Dr Janet Rossant and the extraembryonic lineages during mouse embryogenesis but is not required for reviewers for their valuable criticisms. This study was supported by a migration of primordial germ cells. Development 121, 1487-1496. grant to the University of Wisconsin Medical School under the McLaren, A. (1983). Primordial germ cells in mice. Bibthca anat. 24, 59-66. Howard Hughes Medical Institute Research Resources Program for McLaren, A. (1992). Development of primordial germ cells in the mouse. Andrologia 24, 243-247. Medical Schools, The Graduate School of the University of Noden, D. M. (1989). Embryonic origins and assembly of blood vessels. Am. Wisconsin, and by a University of Wisconsin Hilldale award to third Rev. Respir. Dis. 140, 1097-1103. year undergraduate students (C. H.). 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