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First published online 26 November 2003
doi: 10.1242/dev.00906

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Investigation into a role for the primitive streak in development of the murine allantois

Department of Anatomy, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI 53706, USA

^* Author for correspondence (e-mail: kdowns{at}facstaff.wisc.edu)

Accepted 25 September 2003

	SUMMARY

TOP SUMMARY Introduction Materials and methods Results Discussion REFERENCES

 
Despite its importance as the source of one of three major vascular systems in the mammalian conceptus, little is known about the murine allantois, which will become the umbilical cord of the chorio-allantoic placenta. During gastrulation, the allantois grows into the exocoelomic cavity as a mesodermal extension of the posterior primitive streak. On the basis of morphology, gene expression and/or function, three cell types have been identified in the allantois: an outer layer of mesothelial cells, whose distal portion will become transformed into chorio-adhesive cells, and endothelial cells within the core.

Formation of endothelium and chorio-adhesive cells begins in the distal region of the allantois, farthest from the streak. Over time, endothelium spreads to the proximal allantoic region, whilst the distal outer layer of presumptive mesothelium gradually acquires vascular cell adhesion molecule (VCAM1) and mediates chorio-allantoic union. Intriguingly, the VCAM1 domain does not extend into the proximal allantoic region. How these three allantoic cell types are established is not known, although contact with the chorion has been discounted.

In this study, we have investigated how the allantois differentiates, with the goal of discriminating between extrinsic mechanisms involving the primitive streak and an intrinsic role for the allantois itself. Exploiting previous observations that the streak contributes mesoderm to the allantois throughout the latter's early development, microsurgery was used to remove allantoises at ten developmental stages. Subsequent whole embryo culture of operated conceptuses resulted in the formation of regenerated allantoises at all time points. Aside from being generally shorter than normal, none of the regenerates exhibited abnormal differentiation or inappropriate cell relationships. Rather, all of them resembled intact allantoises by morphological, molecular and functional criteria. Moreover, fate mapping adjacent yolk sac and amniotic mesoderm revealed that these tissues and their associated bone morphogenetic protein 4 (BMP4) did not contribute to restoration of allantoic outgrowth and differentiation during allantoic regeneration.

Thus, on the basis of these observations, we conclude that specification of allantoic endothelium, mesothelium and chorio-adhesive cells does not occur by a streak-related mechanism during the time that proximal epiblast travels through it and is transformed into allantoic mesoderm. Rather, all three cell-types are established by mechanisms intrinsic to the allantois, and possibly include roles for cell age and cell position. However, although chorio-adhesive cells were not specified within the streak, we discovered that the streak nonetheless plays a role in establishing VCAM1's expression domain, which typically began and was thereafter maintained at a defined distance from the primitive streak. When allantoises were removed from contact with the streak, normally VCAM1-negative proximal allantoic regions acquired VCAM1. These results suggested that the streak suppresses formation of chorio-adhesive cells in allantoic mesoderm closest to it.

Together with previous results, findings presented here suggest a model of differentiation of allantoic mesoderm that invokes intrinsic and extrinsic mechanisms, all of which appear to be activated once the allantoic bud has formed.

Key words: Allantois, BMP4, Chorio-allantoic union, Cytokeratins, Differentiation, Endothelium, Gastrulation, Mesoderm, Mesothelium, Mouse, Placenta, Primitive streak, Regeneration, Umbilical cord, Vasculogenesis, VCAM1

	Introduction

TOP SUMMARY Introduction Materials and methods Results Discussion REFERENCES

 
A major goal in developmental studies is to discover how embryonic cells differentiate into a vast array of cell types. In the mouse, it is thought that many critical decisions take place just before and during gastrulation, when the primary germ layers are established (Beddington, 1983).

Pre-eminent in germ layer formation is the primitive streak, a localized thickening in the midline of the epiblast (Bonnevie, 1950; Batten and Haar, 1979). Appearance of the streak defines the posterior end of the future fetus and thus, its anteroposterior axis. The streak is also thought to be where epiblast is transformed into endoderm and mesoderm, both of which are then directed to appropriate sites in the conceptus (Jolly and Ferester-Tadié, 1936; Snell and Stevens, 1966; Poelmann, 1981; Tam and Beddington, 1987; Lawson et al., 1991; Kinder et al., 1999).

Results of both tissue (Beddington, 1982; Copp et al., 1986; Tam and Beddington, 1987) and clonal (Lawson et al., 1991; Lawson and Pedersen, 1992) fate mapping experiments demonstrated that proximal epiblast, located at the embryonic/extraembryonic junction, ingresses into the nascent posterior streak, where it de-epithelializes to emerge as mesoderm (Batten and Haar, 1979; Bellairs, 1986). A large fraction of this mesoderm will be displaced into the extraembryonic region, and lines the exocoelomic cavity (Bonnevie, 1950; Lawson et al., 1991; Lawson and Pedersen, 1992). Shortly thereafter, additional extraembryonic mesoderm accumulates at the posterior angle between the amnion and yolk sac and becomes the allantoic bud (Bonnevie, 1950; Snell and Stevens, 1966) (reviewed by Downs, 1998). Thus, the posterior primitive streak is the allantois' immediate site of origin.

The allantoic bud enlarges into the exocoelomic cavity by a combination of proliferation (Ellington, 1985; Downs and Bertler, 2000), continuous deposition of mesoderm from the streak (Tam and Beddington, 1987; Downs and Bertler, 2000), and distal cavitation (Ellington, 1985; Brown and Papaioannou, 1993; Downs, 2002). During enlargement, two morphologically distinct cell populations are established within the allantois (Ellington, 1985; Downs et al., 1998): an outer layer of `mesothelium', and an inner core of vascularizing mesoderm, defined initially by a plexus of endothelial cells. Formation of the endothelium occurs de novo within the allantois, beginning in its distal region with the appearance of FLK1 (KDR – Mouse Genome Informatics)-containing angioblasts (Downs et al., 1998). Specification of angioblasts and their morphogenesis into endothelial tubules then proceeds proximally to the base of the allantois, where nascent allantoic blood vessels amalgamate with those of the yolk sac and the fetus to create a vascular continuum throughout the conceptus (Downs et al., 1998). Ultimately, the allantoic vascular plexus will be remodeled into an umbilical artery and vein (Kaufman, 1992).

Although the timing of appearance of the mesothelium is not known, it has been proposed, on the basis of limited light microscopic data, that distalmost allantoic cells begin to flatten at approximately the neural plate stage (Downs et al., 1998). By 4-somite pairs, the allantoic projection is enveloped in a morphologically distinct mesothelium (Downs et al., 1998), the distal portion of which contains vascular cell adhesion molecule (VCAM1) (Gurtner et al., 1995; Kwee et al., 1995; Downs et al., 2001; Downs, 2002), required for chorioallantoic union (Gurtner et al., 1995; Kwee et al., 1995). Aside from VCAM1, the only other gene whose protein product has been clearly demonstrated in outer allantoic cells at all stages examined is Ahnak (Kingsley et al., 2001; Downs et al., 2002), although its significance there is not yet known.

On the basis of its ultimate morphology and function, the allantois must eventually contain at least two other cell types: vascular smooth muscle (Takahashi et al., 1996), common to all major arteries and veins, and mesenchymal cells that will provide a connective tissue matrix for the umbilical blood vessels. However, neither of these cell types has been identified in the developing allantois, nor is it known whether they differentiate from allantoic mesoderm or are contributed to the allantois by the chorion after these two tissues unite.

Intriguingly, unlike yolk sac mesoderm, whose transformation into blood islands is dependent upon contact with adjacent endoderm (Wilt, 1965; Miura and Wilt, 1970; Belaoussoff et al., 1998), allantoic mesoderm grows into the exocoelomic cavity as a physically isolated projection. It complexes with the chorion only after allantoic endothelium, mesothelium and chorio-adhesive cells have appeared (Downs and Gardner, 1995; Downs et al., 1998; Downs, 2002).

Thus, given the allantois' relative independence in the exocoelom, it was not clear how the allantois might differentiate. We envisioned at least two strategies. First, the allantois itself might contain intrinsic cues that direct its own differentiation. Second, as the posterior primitive streak is both the site of production of allantoic mesoderm and also initially continuous with it, we postulated that the streak might be integral to allantoic differentiation, specifying allantoic mesoderm as it formed therein, and/or once the allantoic bud had appeared.

Results of several studies support roles in differentiation for both of these tissues. When distal allantoic halves were placed into the exocoelomic cavity of synchronous hosts, donor allantoises united with the hosts' chorion at the appropriate time, despite lack of continuity with the proximal allantoic region and the posterior primitive streak (Downs and Gardner, 1995). Moreover, when allantoises were explanted and cultured in isolation, they invariably vascularized (Downs and Harmann, 1997; Downs et al., 1998) in a stereotypic distal-to-proximal sequence (Downs et al., 2001). Together, these observations suggested a mechanism of differentiation intrinsic to the allantois.

That the streak participates in differentiation of allantoic mesoderm was anticipated in at least two ways, each supported by experimental analysis. In the first, results of heterotopic transplantations suggested that the streak might specify proximal epiblast into particular allantoic cell types while it was being transformed into extraembryonic mesoderm. When nascent posterior mesoderm, the immediate precursor of extraembryonic mesoderm, including the allantois (Tam and Beddington, 1987; Lawson et al., 1991; Kinder et al., 1999), was removed from the primitive streak and transplanted to the distal region of hosts, it exhibited limited developmental potential, failing to colonize the somites of the ectopic site (Dunwoodie and Beddington, 2002). Moreover, when proximal allantoic mesoderm, having just emerged from the streak, was transplanted into a similar ectopic region (Downs and Harmann, 1997), it contributed only to blood vessels, and not to somitic mesoderm. Together, these results suggested that allantoic endothelial cells might be specified within the streak.

Results of a second set of observations suggested that the streak might influence allantoic differentiation after the bud had formed, for example, through factors emanating from a localized signaling center. Allantoic cells closest to the streak might remain initially undifferentiated, whereas those cells farther away would escape the streak's influence and differentiate [proposed by Downs and Harmann (Downs and Harmann, 1997)]. This possibility is consistent with the observation that differentiation of the allantois into endothelium and chorio-adhesive cells begins in the allantois' distal region (Downs and Harmann, 1997); although endothelium is eventually observed throughout the allantois (Downs et al., 1998), VCAM1's domain is restricted to the distal region at all time points examined (Gurtner et al., 1995; Kwee et al., 1995; Downs et al., 2001; Downs, 2002). Thus, distance from the primitive streak might dictate where chorio-adhesive cells arise and are maintained.

Results of several studies have suggested that the posterior streak is indeed a site within which decisions are made that specify cell differentiation and fate, distinguishing extraembryonic from embryonic mesoderm and the future germ line. Intriguingly, the protein products of some homeobox genes, i.e. those transcription factors critical in the regulation of cell proliferation, differentiation, migration, organogenesis and pattern formation during embryogenesis (reviewed by Deschamps and Meijlink, 1992), appear to be involved, as described below.

First, that extraembryonic and embryonic mesoderm may be distinguished in the streak was suggested by expression studies of the homeobox gene, Evx1, and by analysis of genetic knockouts of the Polycomb-group homeobox gene, embryonic ectoderm development, or Eed. Throughout gastrulation, localization of Evx1 was limited to cells near and within the streak (Dush and Martin, 1992). Together with results of fate-mapping, which demonstrated that different types of mesoderm emerge from different axial levels of the streak (Tam and Beddington, 1987; Lawson et al., 1991; Lawson and Pedersen, 1992; Kinder et al., 1999), it was suggested that Evx1 might play a role in establishing the downstream regulatory cascade required for specification of mesodermal cell fate. In a separate study, absence of the Eed gene product led to overproduction of extraembryonic mesoderm, whereas embryonic axial and paraxial mesoderm were missing in the mutants (Schumacher et al., 1996). Together, these observations support the notion that the posterior streak is a region where mesodermal type is specified.

Second, in addition to distinguishing extraembryonic from embryonic mesoderm, the posterior primitive streak is also the site where primordial germ cells (PGCs) are allocated (Chiquoine, 1954; Ozdzenski, 1967; Copp et al., 1986; Ginsburg et al., 1990; Lawson and Hage, 1994; Lawson et al., 1999; Saitou et al., 2002). A recent model has proposed that localized cell signaling within the posterior streak distinguishes extraembryonic mesoderm from nascent germ cells (Saitou et al., 2002). The distinction is thought to be achieved through suppression of Hox gene expression in some cells, thereby allowing them to escape a somatic, or extraembryonic mesodermal, cell fate and retain pluripotency, becoming PGCs. However, the precise nature of these signals is not known.

Thus, to discriminate between roles for the allantois and the primitive streak in differentiation of allantoic mesoderm, we exploited several observations. First, microsurgical removal of allantoises during their pre-fusion phase had previously demonstrated that the streak continuously contributes mesoderm to the allantois, as reflected in the formation of allantoic regenerates over a 20-hour time period (Downs and Bertler, 2000). We reasoned that, if allantoic cell types were specified within the streak, one or more of them might be missing in the regenerates. Alternatively, if the regenerates appeared relatively normal, then we might conclude that differentiation occurred by cues intrinsic to the allantois. A combination of experimental approaches could be used to test this: morphology and the presence of FLK1 within the regenerates' core would identify endothelial cells; morphology and localization of cytokeratins to the outer surface of the regenerates might identify mesothelium; and the functional chorio-allantoic union assay (Downs and Gardner, 1995) together with VCAM1 in outer distal cells of the regenerates would indicate the presence of chorio-adhesive cells (Downs et al., 1998; Downs, 2002). Second, regional specificity of VCAM1 might be established by the streak once the allantoic bud had formed. To test this, whole allantoises could be microsurgically removed and their polarity reversed within the exocoelom. Loss of contact with the streak might induce proximal mis-expression of Vcam1, thereby identifying a role for the streak in localizing chorio-adhesive cells to the distal allantoic region.

Results of this study provide evidence that specification of endothelium, mesothelium and chorio-adhesive cells does not occur within the streak. Nevertheless, they do support a role for the streak in establishment, and possibly maintenance, of the Vcam1 expression domain once the bud has formed. Further, ontogeny of mesothelium does not begin in the distal region, as previously posited (Downs et al., 1998); rather, a variety of junctional complexes are visible throughout the allantoic periphery as soon as the bud appears in the exocoelom.

On the basis of these and previous results, a model of allantoic development is proposed that involves information intrinsic to the allantois, as well as signaling from the primitive streak once the allantoic bud has formed.

	Materials and methods

TOP SUMMARY Introduction Materials and methods Results Discussion REFERENCES

 
Mouse strains, dissection, staging, whole embryo culture
Light-reversed conditions (dark period: 13.00-1.00) maintained all mice used in this study. Matings between F1 hybrids (C57Bl/6xCBA) (Jackson Laboratory) produced an F2 generation that was used as standard `wild-type' material in most experiments. For transplantation experiments, lacZ/+ labeled tissue was obtained by mating F1 hybrid females with ROSA26 males of F1 genetic background and homozygous for two copies of the ubiquitously expressed lacZ transgene (Friedrich and Soriano, 1991). Dissection, staging, whole embryo culture and morphological scoring were previously described (Downs and Davies, 1993; Downs and Gardner, 1995; Downs and Harmann, 1997; Downs et al., 1998; Downs and Bertler, 2000; Downs et al., 2001; Downs, 2002). Most experiments involved conceptuses encompassing all pre-fusion stages, defined from the time of the appearance of the allantoic bud through six-somite pairs, when fusion with the chorion was completed (Downs and Gardner, 1995) (approximately 7.25-8.5 days postcoitum, dpc). In all experiments, operated conceptuses were cultured alongside stage-matched unoperated ones, the latter of which verified culture conditions appropriate for gene expression, endothelialization and chorioallantoic union. Except for experiments involving AlexaFluor594-conjugated Concanavalin A, described below, no significant differences in embryo size or morphological differentiation were noted. After scoring, conceptuses were prepared for histology and immunostaining (Downs and Harmann, 1997; Downs et al., 1998; Downs et al., 2001; Downs, 2002; Downs et al., 2002), described below.

Microsurgical procedures
Creation of allantoic regenerates
Allantoic regenerates were created throughout pre-fusion stages by aspirating whole allantoises through a mouth-held microcapillary after puncturing the anterior yolk sac (Downs and Gardner, 1995; Downs and Bertler, 2000) and culturing operated conceptuses for variable time periods. The base of the allantois had previously been defined as the point of insertion of the allantois into the yolk sac and amniotic mesoderms (Ozdzenski, 1967; Downs and Harmann, 1997). Complete removal of the allantois was verified by visual inspection before culture.

Reversed polarity of whole allantoises
For reversed polarity experiments, whole lacZ/+ donor allantoises were introduced into wild-type host conceptuses whose own allantoises had been removed by aspiration. Where control donor allantoises were placed in normal orientation within the hosts' exocoelom (i.e. distal tips toward the chorion), their proximal ends were labeled by brief (10 seconds) immersion in DiI'/DiIC18(3) (1,1'-dioctadecyl-3,3,3'-tetramethyindocarbocyanine perchlorate, MW 933.88, Molecular Probes, Eugene, OR, USA: D-282) as previously described (Downs et al., 2001). Where donor allantoises were introduced into the hosts' exocoelom with reversed polarity (i.e. proximal ends toward the chorion), their tips, rather than their proximal ends, were labeled. Orientation of donor allantoises was monitored immediately before, during and at the end of the culture period either in the light microscope, as the dye was visible as a pink color on the surface of the cells, or in the inverted compound microscope with brief rhodamine excitation (G2-A filter cube, excitation 535/50, emission 590; Chroma, Rockingham, VT, USA); proximodistal orientation was scored with respect to the anteroposterior and left-right body coordinates. In some experiments, wild-type allantoic tissue confirmed results obtained with lacZ/+ allantoises, as X-gal could obscure the immunostain in the latter.

Isolation and culture of allantoic subregions
Allantoic subregions were excised from whole allantoises at all prefusion stages with the aid of glass scalpels (Beddington, 1987) and either introduced into the exocoelom of host conceptuses whose own allantois had been removed, or cultured in isolation either directly onto tissue culture plastic or free-floating, as previously described (Downs and Harmann, 1997; Downs et al., 1998; Downs et al., 2001).

X-gal- and immunostaining
X-gal staining identified lacZ/+ tissue after fixation in 4% paraformaldehyde, as previously described (Downs and Harmann, 1997; Downs et al., 1998; Downs et al., 2001; Downs, 2002), with the exception that exposure to X-gal was for 2 or 6 hours. Bouin's fluid was used to fix all conceptuses that did not involve X-gal staining. All fixed material was embedded in paraffin wax and sectioned at a thickness of 6 µm. All intact allantoic profiles presented in this study are sagittal views.

Antibodies against FLK1, VCAM1, BMP4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and cytokeratins (DAKO Corporation, Carpinteria, CA, USA) were used in indirect immunohistochemistry. As the literature provided no guidance concerning expression of specific cytokeratins in the allantois, antibodies against a wide spectrum were used. Antibody binding was performed for variable times at room temperature or 4°C, and ready-to-use reagents detected the antibody-antigen complexes (streptavidin-horseradish peroxidase, Vector Laboratories, Burlingame, CA, USA; diaminobenzoate (DAB), DAKO). Pre-binding control peptides to anti-FLK1 and anti-VCAM1 served as negative controls and were previously described (Downs et al., 1998; Downs et al., 2001; Downs, 2002); increasing concentrations of control peptide to anti-BMP4 (Santa Cruz Biotechnology), which was raised against the N-terminal region of the protein, confirmed antibody specificity. Histological sections were also incubated in the absence of antibody in all immunolocalization experiments, including those for cytokeratins for which no control peptide was commercially available. However, antibodies against cytokeratins strongly reacted against trophoblast giant cells, in agreement with previous findings (Hashido et al., 1991; Jaquemar et al., 2003), thereby providing an internal control for antibody specificity. X-gal-stained material was counterstained in nuclear fast red; most other material was stained either in hematoxylin/eosin (H/E) or hematoxylin alone.

Concanavalin A
Lectin-conjugated compounds have previously been used to fate-map cells which border a cavity (Tam and Beddington, 1987). After a brief microspin to remove particulate, AlexaFluor594-conjugated Concanavalin A (absorbance, 590 nm; emission, 617 nm; 2.5-5 mg/ml sterile PBS; Molecular Probes) was gently blown into the exocoelomic cavity of pre-fusion conceptuses via a mouth-held microcapillary in eight separate experiments. After 1 minute of exposure, labeled exocoeloms were repeatedly rinsed with the aid of a mouth-held microcapillary for 3-5 minutes, after which allantoises were removed. ConA labeling of both the mesoderm lining the exocoelomic cavity and the entire surface of isolated allantoises was confirmed (G2-A filter cube, described above, mercury lamp had logged 0-100 hours). After 8-12 or 20-24 hours of culture, allantoic regenerates and unoperated labeled and unlabeled control allantoises were examined by peeling away yolk sacs, cutting out allantoises with a glass scalpel, and viewing them, the yolk sacs and the amnions in both whole-mount and squashed preparations under fluorescence.

To confirm that labeled cells both proliferated and contributed label to their progeny, ConA-labeled headfold- and early somite-stage yolk sacs were isolated, rinsed and treated with a mixture of trypsin and pancreatin (Downs and Harmann, 1997) for 2 minutes on ice, after which the enzyme was inactivated in dissection medium. The mesodermal and endodermal cell layers were then separated via aspiration through a small diameter microcapillary (Beddington, 1987). The cell layers were placed separately into sterile Eppendorf tubes, and further enzyme-treated (15 minutes on ice) to obtain single cell suspensions, which were briefly spun down, resuspended in dissection medium, and plated onto 8-well chamber slides (Nunc Permanox; Nunc, Naperville, IL, USA) containing 0.4 ml culture medium. After 5 hours, floating cells were removed, the adherent cells were fed, counted and cultured for 20 hours more, at which time the medium was again replaced, and the adherent labeled cells counted. In addition, we verified that AlexaFluor ConA did not inhibit cell motility by plating labeled aspirated allantoises (two experiments, n=10) directly onto tissue culture plastic (Downs et al., 2001) and demonstrating the presence of labeled mesothelial outgrowth at 8 and subsequently 20 hours.

In eight experiments, all 21 unlabeled, unoperated conceptuses were normal at the end of the culture period and negative for ConA. Of 27 control conceptuses whose allantois had been labeled but not removed from the exocoelom, one fetus (3.7%) was abnormal in that the ConA-labeled allantois had fused with the yolk sac rather than with the chorion. Of the 72 conceptuses whose allantoises had been labeled, removed and subsequently regenerated, none were abnormal, although one conceptus (1.4%) was dead; a subset of these (n=22) was measured in the dissection microscope (see next section) and their average length compared with that of unlabeled regenerated allantoises (n=6). In a final set of control experiments, allantoises were removed prior to labeling exocoelomic cavities (two experiments, eight conceptuses, two fetuses of which were abnormal after culture) to ascertain the probable labeling pattern if yolk sac/amniotic mesodermal cells crawled over the primitive streak during regeneration, as well as to verify persistence of label in the regenerates.

Measurement of allantoises and the VCAM1-free region
The length of some 20- and 6-hour allantoic regenerates and unoperated allantoises was measured in the dissection microscope immediately after culture, by means of an eyepiece reticule, and plotted (Fig. 2A,B) or reported in Results. The average length and standard error of the mean (s.e.m.) of histologically prepared and sectioned allantoises and their VCAM1-negative regions were obtained from the longest three sagittally oriented sections after photography, computer scanning (Adobe Photoshop) and printing (Fig. 4).

View larger version (51K): [in this window] [in a new window]   Fig. 2. Comparison of allantoic lengths in 20-and 6-hour regenerates and unoperated controls. Bar graphs of the length (in µm) of (A) unfused and fused allantoic regenerates, and control unoperated allantoises after 20 hours in culture, and (B) allantoic regenerates and control unoperated allantoises after 6 hours in culture, measured in the dissection microscope. Allantoic lengths were plotted against the developmental stage at which the culture period began. In B, the single regenerate created at 6-somite pairs was so small that it could not be accurately measured in the dissection microscope; thus, no bar is seen at this time point. The total number of allantoises scored in each category is indicated at the base of each column of the graph; vertical lines at the apex indicate the s.e.m.s; and asterisks in A indicate significant differences in length between unfused allantoic regenerates and unoperated control allantoises for each time point (P<0.05). Abbreviations: EB, neural plate/early allantoic bud stage; LB, neural plate/late allantoic bud stage; EHF, early headfold stage; LHF, late headfold stage; 1-6-s, 1- to 6-somite pairs.

View larger version (55K): [in this window] [in a new window]   Fig. 4. The VCAM1-negative proximal region is approximately 220 µm long. The average total lengths (in µm) of allantoises and the VCAM1-negative proximal region from fixed ex vivo conceptuses were plotted as a function of developmental stage of the embryo. The number of allantoises examined at each time point is provided in the base of each bar; vertical lines at the top of each bar represent the s.e.m. Bars with only one specimen (9-s, 11-s) nonetheless contain errors, as measurements were the average of three histological sections for each allantois (see Materials and methods). After 6-somite pairs, the length of the VCAM1-negative region only is indicated because representation of the complete profile of fused allantoises was difficult to achieve in histological sections.

Statistical analyses
The Student's two-way t-test (Mini-Tab, equal variances assumed, Confidence Interval=95%) determined significant differences (P<0.05) between treatment categories wherever relevant in this study.

	Results

TOP SUMMARY Introduction Materials and methods Results Discussion REFERENCES

 
The primitive streak is active during pre-fusion allantoic development
Results of earlier studies suggested that allantoises microsurgically removed from the conceptus prior to their union with the chorion would regenerate (Downs and Gardner, 1995). Later, allantoic regeneration was exploited to determine when and how much mesoderm was added to the allantois by the streak (Downs and Bertler, 2000). Findings revealed streak activity at all pre-fusion stages, although mesoderm contribution decreased with increasing developmental age. On the basis of these observations, we hypothesized that allantoic regenerates would be a powerful experimental means by which to investigate the effect of streak activity on differentiation of allantoic mesoderm. The rationale was that, if any of the regenerates lacked certain cell populations, then specification of the missing cell type(s) might have occurred within the streak at or slightly later than the time of allantoic removal.

To ensure that regenerates formed at all pre-fusion stages, whole allantoises were removed, exposing the subjacent posterior primitive streak (Fig. 1A-E). After 20 hours in culture, operated conceptuses were examined in the dissection microscope and regenerates were measured. Regeneration was found at all developmental stages, but only a few regenerates grew far enough to unite with the chorion (Fig. 2A). The average length of `unfused' regenerates was remarkably similar between the neural plate and 5-somite pair stages (501.5±21.7 µm, n=54), but significantly shorter (P<0.000) than unoperated controls (924.7±17.6 µm, n=44). The average length of regenerates formed from the 6-somite pair stage was significantly shorter (244.6±66 µm, n=6) than the other regenerates (P=0.002, Student's two-way t-test with 5-somite pair regenerates). Thus, on the basis of these findings, we conclude that, during pre-chorionic fusion stages, the primitive streak is active at all time points, producing regenerated allantoises.

View larger version (73K): [in this window] [in a new window]   Fig. 1. Morphology and immunostaining in 20-hour allantoic regenerates. (A-C) Schematic drawings illustrate the formation of allantoic regenerates between the neural plate/early allantoic bud (EB) through 6-somite pair stages (lower left). (A-B) The allantois (al) overlies the primitive streak (ps) and was removed. (B) Epiblast (curved red arrow) continues to ingress into the primitive streak, so that, during whole embryo culture, operated conceptuses regenerate a new allantois (al-r) (C). (D,E) H/E-stained low- (D) and high- (E) magnification views of the posterior primitive streak immediately after removal of the allantois. The arrow in D indicates the anterior point of microcapillary entry into the yolk sac. Arrowheads in D and E indicate the site of removal of the allantois, just above the primitive streak. (F) H/E-stained 20-hour allantoic regenerate (al-r) contains mesothelium (m) and nascent endothelial channels (asterisks). (G-L) Immunostaining of allantoic regenerates and cultured unoperated controls. Histological sections immunostained with one antibody (G,H,J,K) were counterstained in hematoxylin. Doubly immunostained histological sections (I,L) were not counterstained. (G) FLK1 (brown color) in the endothelial plexus (e.g. asterisks) throughout the allantoic regenerate. (H) VCAM1 (brown color) in the mesothelium (m) of the distal allantoic two-thirds and a few distal core cells (arrowhead) of the regenerate. (I) FLK1 (blue color)/VCAM1 (brown color) double immunostaining reveals closely associated but separate populations of FLK1- and VCAM1-containing cells within the distal core, as previously reported (Downs et al., 2001). (J-L) Control unoperated host allantoises immunostained for (J) FLK1, (K) VCAM1 and (L) FLK1 (blue color)/VCAM1 (brown color). Other abbreviations: ac, amniotic cavity; am, amnion; ch, chorion; ec, ectoplacental cavity; x, exocoelomic cavity; ys, yolk sac. In F-L, embryo stages before and after culture are separated by `/'. Scale bars in L: 50 µm (E); 100 µm (F,G,I); 200 µm (D,H,J-L).

FLK1 was found in the endothelial cells of 20-hour regenerates, and never in the mesothelium (Fig. 1G) (Downs et al., 1998). Similarly, VCAM1 was appropriately localized to the distal two-thirds of all allantoic regenerates, which included the mesothelium and some core cells (Fig. 1H) (Downs et al., 2001; Downs, 2002). Double immunohistochemistry revealed normal cell-cell relationships in the regenerates: VCAM1-positive cells were associated with, but distinct from, the FLK1-positive core vasculature, and absent from the proximal allantoic third (Fig. 1I-L) (Downs, 2002).

Of the 77 allantoic regenerates of Fig. 2A, a small number (22%) had undergone union with the chorion. In these, microscopic inspection revealed that the early steps of placentation were occurring: the chorio-allantoic fusion surfaces appeared to be breaking down, and the FLK1-positive allantoic vasculature was penetrating chorionic ectoderm (data not shown), as previously described (Downs, 2002). Of the remaining regenerates, none grew far enough to unite with the chorion. To discover whether these were nonetheless functionally normal, lacZ/+ unfused regenerates were aspirated at 20 hours, placed into the exocoelom of pre-fusion wild-type host conceptuses whose own allantoises had been microsurgically removed, and the operated hosts were cultured to just beyond 6-7-somite pairs, when all allantoises have normally united with the chorion (Downs and Gardner, 1995; Downs, 2002). In two experiments, 4/4 grafted X-gal-stained regenerated allantoises exhibited convincing union with the hosts' chorion (data not shown), suggesting functional adhesive capabilities.

Why some regenerates grew far enough to unite with the chorion could be explained by prolonged collapse of the exocoelomic cavity, possibly because the yolk sac puncture did not heal in a timely manner. As a consequence of reduced exocoelomic volume, they could have been brought into closer proximity to the chorion, thereby initiating union.

Thus, not only do regenerates demonstrate appropriate morphological and molecular properties, but they are also functionally competent, capable of uniting with the chorion if given the opportunity.

Formation of mesothelium
The normal morphological, molecular and functional profiles of 20-hour regenerates suggested that the primitive streak does not specify allantoic mesoderm prior to its becoming the allantoic bud. Although outer cell morphology and expression of Vcam1 alluded to the presence of mesothelium in the regenerates, flattening of distal outer cells cited as a provisional criterion for the onset of formation of mesothelium at the neural plate stage (Downs et al., 1998), was subjective and non-rigorous. Thus, electron microscopic analysis was used here to determine when and where junctional contacts, indicative of an epithelium, were present on the outer surface of the allantois.

The number of contacts between outer cells was scored in ultrathin sections of allantoises spanning the neural plate/early bud through 4-somite stages (Table 1). Three types of electron dense structures, including spot desmosomes, adhesive plaques and presumptive tight junctions (Batten and Haar, 1979), were identified at sites of contact at all stages (Fig. 3A-D), with neither the distal nor proximal halves favored (Table 1). Moreover, these structures were not always found in flattened cells. Finally, at the neural plate and headfold stages, the only junctional contacts observed in cells of the core were with outer cells (Fig. 3A), suggesting that the otherwise junction-free nascent core mesodermal cells shared ultrastructural similarities with migrating mesoderm rather than with epithelium (Batten and Haar, 1979).

View larger version (106K): [in this window] [in a new window]   Fig. 3. Presence of mesothelium as detected by transmission electron microscopy and cytokeratins. (A-D) Transmission electron micrographs reveal various structural components of the plasma membrane at sites of cell-cell contact in outer cells of the allantois. (A) Ultrathin section through two outer cells (1,2), and an inner core cell (3) in the proximal region of an early allantoic bud (EB). The arrow points to a desmosome between the two outer cells, whereas the arrowheads indicate electron densities, possibly adhesion plaques or glancing sections through desmosomes, at sites of contact between the outer cells and an inner cell. Scale bar: 500 nm. (B) Ultrathin section contains a possible junctional complex between two cells (1,2) in the proximal region of an early allantoic bud (EB), consisting of a putative tight junction (left of asterisk), adhesion plaque (arrowhead), and spot desmosome (arrow). Scale bar: 500 nm. (C) Ultrathin section through the proximal region of an EHF stage allantois shows an isolated adhesion plaque very similar to those previously described (Batten and Haar, 1979). Scale bar: 100 nm. (D) Ultrathin section through the distal region of a 4-somite pair (4-s) allantois contains an apparently mature spot desmosome between two outer cells. Scale bar: 100 nm. (E-G) Brightfield photomicrographs exhibit immunostaining against cytokeratins (brown color, arrows) in nascent allantoises of ex vivo specimens. Sections were counterstained in hematoxylin. (E) EB stage. (F) LB stage. (G) EHF stage. Other abbreviations as in Fig. 1. Scale bar in G: 50 µm (E-G).

Morphological and molecular differentiation in allantoic regenerates occurs with normal kinetics
To verify that differentiation took place with normal kinetics in allantoises post-microsurgical removal, 6-hour regenerates were created and analyzed for morphology and gene expression. As a prelude to this analysis, we first established the distance from the primitive streak at which VCAM1 appeared in ex vivo conceptuses (Fig. 4). VCAM1 was visible in distal mesothelial cells only when allantoises were at least 220.7 µm (±5.5 µm) long (data not shown; 1-somite pair; n=4). At 1-somite pair and all stages thereafter, the average length of the VCAM1-negative region was fixed at approximately 220.2 µm (±5.9 µm, n=34) (Fig. 4). This length did not differ significantly in conceptuses cultured for 20 hours (193.1±16.5 µm, n=9; P=0.08; data not shown), confirming that culture conditions did not alter correct topographical expression of Vcam1. The length of the VCAM1-negative region in fused regenerates was 192.4 µm (±12.8 µm; n=12; data not shown), similar to the unoperated cultured controls, whereas in unfused regenerates it was 149.5 µm (±8.0 µm; n=14; data not shown), significantly shorter than the controls (P=0.01). This difference may be attributable to the fact that once allantoises are anchored to the chorion, they may `stretch' during subsequent enlargement of the exocoelomic cavity and lengthen; in the absence of anchoring to the chorion, `stretching' would not occur in the unfused regenerates. Thus, after long-term culture, unfused regenerates might ultimately appear slightly collapsed and shorter.

In the next set of experiments, 6-hour regenerates were created (Fig. 2B, Fig. 5). Their average length before fixation was 177.6±19.2 µm (n=23; Fig. 2B). As we found that Bouin's fluid and subsequent histological processing resulted in tissue shrinkage of 14.8% ±2.2% (n=14 fresh and subsequently fixed specimens, neural plate – 5-somite pair stages; data not shown), gene expression in the 6-hour regenerates was predicted to resemble that found in fixed ex vivo early headfold stages (Fig. 4).

View larger version (132K): [in this window] [in a new window]   Fig. 5. Localization of VCAM1, cytokeratins and FLK1 in 6-hour allantoic regenerates. Allantoises were removed and operated conceptuses were cultured for 6 hours. Embryo stages before and after culture are indicated at the bottom of each panel (A,C-H), and are separated by `/'. Immunostain is brown in all panels; all sections were counterstained in hematoxylin. (A) Allantoic regenerate (al-r), 3/6-somite pairs, VCAM1-immunostained. This regenerate was less than 220 µm long and was negative for VCAM1 (i.e., no brown staining). Arrowhead indicates mesothelium. (B) Internal control, same conceptus as A, confirms the presence of VCAM1-positive cells in the heart (surrounding the asterisk) (Gurtner et al., 1995; Kwee et al., 1995). (C) VCAM1-stained unoperated cultured control allantois (al) from the same experiment as the specimen in A,B. (D) Brightfield photomicrograph shows cytokeratins (arrows) in both the allantoic mesothelium and core cells of the 6-hour regenerate (al-r). (E-H) Brightfield photomicrographs show FLK1 in the 6-hour regenerates (E,G) and corresponding unoperated cultured controls (F,H). Other abbreviations as in Fig. 1. Scale bar in H: 50 µm (A,B,D-G); 75 µm (H); 100 µm (C).

The presence of mesothelium was then confirmed in a second set of 6-hour regenerates by antibody staining against cytokeratins (Fig. 5D). As the average length of fixed 6-hour regenerates was similar to normal headfold-stage allantoises, described above, it was therefore not unexpected that cytokeratins were found in the regenerates' outer and inner cell populations, as reported in the previous section for expression of cytokeratins in ex vivo headfold-stage allantoises (Fig. 3G).

FLK1, which was previously observed in the distal core allantoic region between the late bud/neural plate and 2-somite pair stages (Downs et al., 1998), also showed distal staining in 6-hour regenerates created between the neural plate- and 2-somite pair stages; the primitive streak was also appropriately negative (Fig. 5E,F) (Downs et al., 1998). Not surprisingly, those regenerates created after 2-somite pairs did not exhibit regional polarity of FLK1 (Fig. 5G,H), as it was known that, by the time of microsurgery, the primitive streak region had become FLK1-positive, thereby making distal polarity of Flk1 expression in these later regenerates impossible to discern (Downs et al., 1998).

Thus, taken together, our results revealed that allantoic regenerates behave like intact allantoises, no matter when they are formed: mesothelium was present, VCAM1 identified the chorio-adhesive cell type at a fixed and reproducible distance from the primitive streak, and endothelial cell formation began in the distal region during regeneration. On the basis of these observations, we conclude that specification of mesothelial, chorio-adhesive and endothelial cells does not occur within the primitive streak.

Differentiation within allantoic regenerates does not involve healing by yolk sac and amniotic mesoderm
Although the aforementioned observations provide compelling evidence that information intrinsic to the allantoic bud is responsible for re-establishing appropriate differentiation in the regenerates, an equally plausible mechanism could involve the adjacent yolk sac and/or amnion. Specifically, we hypothesized that, after removal of the allantois, the exposed primitive streak `heals' by contribution of mesoderm from the yolk sac and amnion. The allantoic regenerate would then grow into and become coated in cells of yolk sac and/or amniotic origin (Fig. 6), which would then reorganize allantoic development. The molecular cue responsible could be the gene product of BMP4 as, in its absence, allantoises do not form or they are highly reduced in size and abnormal in morphology (Winnier et al., 1995; Lawson et al., 1999; Fujiwara et al., 2001). Moreover, both the yolk sac and amnion contain BMP4 (Lawson et al., 1999).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Hypothetical model of growth of the allantoic bud and regenerates into BMP4-positive mesoderm of the exocoelomic cavity. Schematic diagram hypothesizes how growth of the allantois and allantoic regenerates might occur by a `finger-in-a-glove' mechanism (suggested by Downs) (Downs, 1998). (A) Normal (non-hypothetical) neural plate/No bud (0B) stage, approximately 7.25 dpc. Thick lines (both black and red) are BMP4-positive with black color the mesodermal component of the yolk sac and amnion, and the red color the future site of the allantoic bud. The localization profile of BMP4 at this stage is from Lawson et al. (Lawson et al., 1999). (B) In this hypothetical scenario, the normal allantois grows into the BMP4-positive corner like a finger-in-a-glove, the result of which is an allantoic bud whose outer surface contains BMP4-positive cells. (C) The allantois has been microsurgically removed and, in the subsequent hypothetical scenario, BMP4-positive mesoderm from the yolk sac and amnion crawls onto the exposed primitive streak, thereby re-establishing the BMP4 expression pattern of the normal 0B stage in A, and subsequently that of B in the regenerates. Abbreviations as in Fig. 1.

View larger version (121K): [in this window] [in a new window]   Fig. 7. Localization of BMP4 to the extraembryonic region of mouse gastrulae and allantoic regenerates. BMP4 (brown color) in the extraembryonic region of mouse conceptuses in ex vivo (A,B,D,F,G) and cultured unoperated (E,H) allantoises, and allantoic regenerates (I-M). Ex vivo control conceptus [pre-binding with control peptide, CP] (C). See text for complete explanation of staining patterns. In E,H,I, embryo stages before and after culture are separated by `/'. (A) EB stage. BMP4 is present in mesoderm lining the exocoelomic cavity and in the mesothelium surrounding the allantois at this stage, as shown in this glancing sagittal section through the lateral surface of an allantoic bud, in agreement with previous results (Lawson et al., 1999). In deeper sections, the core of the early bud was negative (data not shown). (B) EHF stage. (C) EHF stage, pre-binding antibody with control peptide (+ CP). The arrow indicates faint BMP4 in yolk sac mesothelium, suggesting that this amount of control peptide (100:1, CP:anti-BMP4) was not completely efficacious in pre-binding all antibody, although all other tissues were negative. (D) Two-somite pairs (2-s). (E) Cultured conceptus (EHF/4). (F) Six-somite pairs (6-s). BMP4 is now present throughout the allantois, including endothelium within the core. (G) Sixteen-somite pairs (16-s), BMP4 is present at the chorio-allantoic fusion junction. (H) Cultured conceptus (3/13); BMP4 is present in both mesothelium and in endothelial elements within the core. (I) Twenty-hour allantoic regenerate (3/14) demonstrates a similar BMP4 staining pattern in the allantois as that shown in H. (J-M) Time course of allantoic regenerates created from EHF-stage conceptuses shows BMP4 in the regenerates at (J) 0 hours, (K) 1.5 hours, (L) 3.0 hours and (M) 6.0 hours after allantoic removal. Arrows in J-L indicate the distal site of allantoic regeneration. Abbreviations as in Figs 1, 2. Scale bar in M: 50 µm (A-C,J-M); 100 µm (I); 150 µm (D-G); 200 µm (H).

To address this, the adjacent yolk sac and amniotic mesoderms were fate-mapped by labeling the mesodermal lining of the exocoelomic cavity with AlexaFluor-conjugated Concanavalin A, which selectively binds -mannopyranosyl and -glycopyranosyl residues of glyco-conjugates on the cell surface. First, to verify that ConA-labeled cells proliferated and passed their label on to progeny cells, single cell suspensions of the mesodermal and endodermal components of one of the tissues, the yolk sac, were plated onto tissue culture plastic (see Materials and methods). After 20 hours in culture, endodermal cells remained floating, and did not adhere to the plastic, in agreement with findings by others (R. Gardner, personal communication). In contrast, both labeled and unlabeled yolk sac mesodermal cells adhered to the plastic and had a vacuolated appearance (Fig. 8A-D). In three experiments, AlexaFluor ConA-labeled cells doubled in number over the 20-hour culture period (Table 2). We also confirmed that ConA-labeling did not inhibit mesothelial cell motility, by plating labeled allantoises (Downs et al., 2001) (Fig. 8E) and observing outgrowth at 8 (Fig. 8F) and 20 hours thereafter (data not shown). Moreover, the explants looked normal, in that they were well vascularized by 20 hours and morphologically similar to unlabeled controls, as previously described (Downs et al., 2001). Finally, use of ConA in these circumstances further revealed that allantoic outgrowth was mesothelial in origin.

View larger version (126K): [in this window] [in a new window]   Fig. 8. AlexaFluor594-conjugated Concanavalin A does not affect mesodermal cell proliferation or mesothelial cell migration, and is inherited by daughter cells. Brightfield (A,C-E) and fluorescent (B,F) photomicrographs of AlexaFluor594-ConA-labeled yolk sac mesoderm and allantoic mesothelium. (A,B) ConA-labeled yolk sac mesodermal cells at 20 hours after culture. Arrowhead points to structures which give these cells a vacuolated appearance. (C) Control unlabeled yolk sac mesodermal cells, 20 hours after culture. Arrowhead points to `vacuolated' region, whereas the arrow points to a filopodial projection often seen on both labeled and unlabeled cultured yolk sac mesodermal cells. (D) Example of a very minor cell population found in cultures of both labeled and unlabeled yolk sac mesodermal cells, but which were never labeled, and are thus possibly cells of blood island provenance. (E,F) ConA-labeled headfold-stage allantoic explant at 8 hours of culture. Arrows point to labeled mesothelial outgrowth. In this and Figs 9, 10, 11, d: distal, p: proximal. Scale bar in F: 50 µm (A-D); 100 µm (E,F).

View larger version (57K): [in this window] [in a new window]   Fig. 9. Fate mapping the yolk sac and amniotic mesoderm with AlexaFluor594-conjugated Concanavalin A. Allantoises in D-I are from conceptuses that had been cultured for 20-24 hours and exposed on the same film to compare relative intensities of allantoic fluorescence within the same experiment. Embryo stages before and after culture in D-I are separated by a `/'. All panels contain gross microscopic views. (A-B) Posterior region of a LHF-stage conceptus in brightfield (A) and viewed by fluorescence after (B) labeling the exocoelomic cavity (x) with AlexaFluor ConA. (C) The allantois (al) has been removed from A to show complete superficial ConA labeling by fluorescence. (D,E) Cultured conceptus (4/12) in brightfield before (D) and in corresponding fluorescence (E). That most of the label is in the allantois and not the chorion was revealed by cutting the allantois away from the chorion and re-examining it (not shown). (F,G) Brightfield (F) and corresponding fluorescent (G) regenerated control allantois (EHF/9) in which the exocoelom had been labeled after removal of the allantois, thereby marking the posteriormost level of the streak. Arrowheads provide examples of very small individually labeled cells and patches of positive cells arranged in a lateral line spanning proximal to distal, typical of all six such `pre-labeled' allantoic regenerates. (H,I) Brightfield (H) and corresponding fluorescent (I) images of an allantoic regenerate (2/11) (al-r) that contains two very small positive cells (arrowheads) in the distal region. Scale bar in I: 100 µm (C-I); 400 µm (A,B).

On the basis of these findings, we conclude that labeling the exocoelomic cavity of headfold- and early somite stage conceptuses with ConA has no detrimental affect on embryonic growth, embryonic differentiation or on allantoic regeneration. Although a very small number of yolk sac/amniotic cells may crawl over the exposed primitive streak and become incorporated into the lateral surface of the regenerates, failure to identify them in the majority of regenerates strongly suggests that differentiation in allantoic regenerates is not re-specified by a healing process that involves the adjacent yolk sac, amnion or BMP4 contained therein. Moreover, these results accord with previous ones, which investigated the fate of presumptive PGCs thought to reside in the base of the allantois (Ozdzenski, 1967); results of those studies demonstrated that cell movement at the posterior embryonic/extraembryonic junction is typically away from the streak, rather than toward it, the consequence of which is that re-entry of allantoic primordial germ cells into the fetus probably does not occur (Downs and Harmann, 1997; Anderson et al., 1999).

Proximal allantoic regions become VCAM1-positive following loss of contact with the streak
As described above, the 220 µm-long VCAM1-negative region remained fixed throughout allantoic development, suggesting either that the allantois acquires intrinsic information that limits the domain of VCAM1, or that the streak suppresses VCAM1 in the proximal allantoic region closest to it.

To investigate a role for the streak in suppressing VCAM1, whole donor allantoises were removed and placed into the exocoelomic cavity of hosts, either in normal or reversed orientation (Fig. 10), and cultured for 8 hours. We reasoned that, if the streak suppresses gene expression or translation of VCAM1 transcripts in cells closest to it, then the proximal region would exhibit VCAM1 upon release from contact with the streak.

View larger version (82K): [in this window] [in a new window]   Fig. 10. VCAM1 and FLK1 in isolated whole allantoises and allantoic subregions after 8 hours of culture. Whole allantoises and allantoic subregions were placed into the exocoelomic cavity or in isolation in test tubes and cultured for 8 hours, after which they were VCAM1 (A-G) or FLK1 immunostained (H-J) (brown color). Allantoises from wild-type unoperated cultured (D,G) and/or lacZ/+ ex vivo conceptuses (C) were used as controls. Sections were counterstained in nuclear fast red (A-D,H-J) or hematoxylin (E-G). In A-B, E-F and H-J, stages of synchronous donor allantoises and host conceptuses before and after culture are separated by a `/'. In D,G, stages of whole conceptuses before and after culture are separated by `/'. An ex vivo allantois is contained in C. (A) VCAM1 in distal region of whole X-gal-stained allantois in normal orientation. (B) VCAM1 is maintained in the distal region of a reversed polarity whole X-gal-stained allantois. The VCAM1-negative proximal region has made contact with the chorion. (C) Normal distal VCAM1 in X-gal-stained lacZ/+ ex vivo control conceptus from the same experiment as A,B. (D) Wild-type unoperated cultured conceptus from same experiment as A-C shows normal distal VCAM1 in the allantois. (E-G) Allantoises are from wild-type conceptuses. (E) Proximal allantoic third does not contain VCAM1. (F) Distal two-thirds of the same allantois in E contains VCAM1. (G) Unoperated cultured control conceptus from same experiment as E,F. (H-J) Whole X-gal-stained allantois (H), X-gal-stained distal two-thirds (I), and X-gal-stained proximal third (J) of the same allantois in I exhibit FLK1 throughout the allantoic core. Abbreviations as in Figs 1, 2, 8. Scale bar in J: 50 µm (E,F); 75 µm (A,H-J); 100 µm (B-D,G).

Allantoises were then removed and, based on measurements of the VCAM1-negative region (Fig. 4), cut into distal and proximal subregions. To ensure that the proximal regions fell to within at least one standard deviation of the average 220 µm VCAM1-negative region (data not shown), these were never longer than 150 µm. Proximal and distal allantoic subregions were then placed separately into the exocoelomic cavity of individual hosts, and cultured for either 8 or 24 hours.

All 8-hour proximal regions were negative for VCAM1 (Fig. 10E). Seven out of 24 proximal regions were free-floating in the exocoelom and 3/24 were tethered to the host's regenerated allantois (data not shown); all had formed a complete outer rind of mesothelium (Fig. 10E). Fourteen out of 24 proximal regions had undergone union with the chorion (data not shown), exhibiting mesothelium at the unfused edge and, where they had made contact with the chorion, cells appeared to be breaking down (Downs, 2002). Of eight control distal regions, all