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
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SUMMARY |
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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
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Introduction |
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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.
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Materials and methods |
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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).
|
|
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.
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Results |
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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.
|
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
).
|
|
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).
|
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
).
|
|
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.
|
|
|
|
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.
|
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 were appropriately VCAM1-positive
(Fig. 10F), with levels of
VCAM1 approximating those of the intact controls (compare
Fig. 10F with 10G). Both
8-hour distal and proximal regions were positive for FLK1
(Fig. 10I,J).
By 24 hours, 21/21 distal regions maintained VCAM1, with levels similar to intact allantoises (Fig. 11A). However, in contrast to the 8-hour explants, all 21/21 proximal regions had become VCAM1-positive throughout the mesothelium and core (Fig. 11B,C). One proximal region was tethered to the tip of the host's allantoic regenerate (Fig. 11B), and one had fused with both the chorion and the yolk sac (Fig. 11C). All the others had united with the chorion alone and behaved like distal subregions, with mesothelium breaking down at the fusion junction (data not shown). FLK1 localization was unchanged, being present in both the proximal and distal regions (data not shown). Further support for these observations was obtained by culturing subregions in suspension outside of the conceptus. Ten out of 10 distal and 10/10 proximal regions exhibited VCAM1 (Fig. 11D,E), whereas all 4/4 whole control allantoises were VCAM1-positive in both the outer mesothelial circumference and the core (Fig. 11F).
|
To distinguish between signaling from the primitive streak and/or the allantoic midregion in inhibiting proximal VCAM1, distal allantoic thirds were excised from whole allantoises, maintaining continuity between the mid- and proximal regions (`mid/proximal'). In the same experiments, whole allantoises, distal and mid/proximal regions were cultured individually within the exocoelomic cavity of host conceptuses. As expected, distal thirds were always strongly VCAM1-positive (Fig. 11G). Moreover, not only was VCAM1 found throughout explanted whole allantoises (Fig. 11I), but it was also found throughout the mid/proximal subregions, although only two of these had remained free-floating in the exocoelom (Fig. 11H). Failure of the allantoic midregion to suppress proximal VCAM1 was confirmed in three additional experiments of isolated explants cultured in suspension for the same time period (data not shown).
On the basis of these observations, we conclude that the primitive streak limits the expression domain of VCAM1, confining the chorio-adhesive cell type to the distal allantoic region. In contrast, the streak is neutral with regard to formation of the mesothelium and endothelial-based vasculature.
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Discussion |
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The murine allantois acquires intrinsic information as the allantoic bud forms
That the allantois contains intrinsic factors responsible for its own
differentiation was demonstrated by studying its regeneration. A combination
of morphological and immunohistochemical approaches were used to demonstrate
the normal kinetics of allantoic regeneration, including the invariable
presence of mesothelium as defined by cytokeratins, a FLK1-positive
endothelial plexus, and a defined VCAM1-positive distal region, indicative of
the presence of chorio-adhesive cells. In addition, despite their smaller
size, 20-hour regenerates were found to be competent to unite with the
chorion. Repair of the exposed primitive streak from the adjacent yolk sac and
amnion, possibly stimulated by BMP4 contained therein, was not consistently
involved in allantoic outgrowth or in the re-establishment of proper
morphological and molecular patterning in the regenerates. However, we cannot
discount the possibility that BMP4, which was found in the normal allantoic
bud and all of the regenerates (Fig.
7), may originate within the allantois and thereby contribute to
its differentiation. Moreover, although cell age appears to play a major role
in the timing of differentiation once the bud has formed, as discussed in the
model below, the period during which proximal epiblast is converted into
mesoderm within the streak plays no discernible role in specification of
allantoic cell types.
These observations strongly suggest that, as the allantois becomes visible in the exocoelom, it acquires information that will direct its own differentiation.
The primitive streak establishes VCAM1's expression domain within the allantois
Although the streak does not specify the chorio-adhesive cells, it does
appear to be involved in restricting their domain within the allantois by
suppressing expression of Vcam1 in the proximal allantoic region.
The base of the allantois, which corresponds with the posteriormost level
of the streak, has previously been defined as the point of insertion of the
allantois into the yolk sac and amniotic mesoderm
(Ozdzenski, 1967) (reviewed by
Downs, 1998
). This definition
has guided the present and all previous studies involving microsurgical
removal of the allantois (Downs and
Harmann, 1997
; Downs et al.,
1998
; Downs and Bertler,
2000
; Downs et al.,
2001
; Downs,
2002
). Accordingly, measurements on histologically prepared
allantoises revealed that VCAM1 was consistently localized to the distal
region, beginning at a distance of 220 µm from the posterior limit of the
primitive streak (Fig. 4). In
isolation, proximal regions of allantoises were negative during the first 8
hours of culture, but by 24 hours they contained VCAM1 and behaved like distal
tips in their ability to unite with the chorion
(Fig. 11). Co-culture
experiments revealed that suppression of Vcam1 expression in the
proximal region of the allantois was not because of its being in contact with
its mid-region (Fig. 11).
On the basis of these data, we conclude that, although the primitive streak does not specify chorio-adhesive cells during formation of allantoic mesoderm, it does suppress VCAM1, either at the level of transcription or translation, in the proximal allantoic region once the bud has formed. Whether the mechanism of suppression involves direct cell contact and/or localized signaling from the streak remains to be determined.
Formation of allantoic mesothelium
On the basis of limited light microscopic data, we had previously supposed
that formation of mesothelium began in the distal allantoic region
(Downs et al., 1998). Thus, an
unexpected finding in this study was the presence of this layer as soon as the
bud appeared (Fig. 3).
Moreover, mesothelium was found in all of the regenerates, as well as in
explanted whole allantoises and allantoic subregions. These observations argue
that mesothelium forms as a result of cues intrinsic to the allantois.
What those cues are is unknown, although it is tempting to speculate that
they involve cells being on an outer surface. In other words, differentiation
of mesothelium may share similarities with that of trophectoderm of the
blastocyst, where differentiation of blastomeres along this lineage seems to
depend upon being located externally
(Tarkowski and Wroblewska,
1967). Thus, mesothelium of the allantois may differentiate by an
inside-outside mechanism, as a result of formation of asymmetrical cell
contacts between outer and inner cells. Indeed, in our limited study, outer
and immediately subjacent inner cells were linked by junctional structures
(Fig. 3A) that were not found
anywhere else in the allantoic core during early stages. Intriguingly, the
giant phosphoprotein, Ahnak, which is found near desmosomal contacts on
epithelial cells (Hashimoto et al.,
1995
; Masunaga et al.,
1995
), was recently described on the allantoic surface
(Kingsley et al., 2001
). Ahnak
was present there as early as the neural plate stage
(Downs et al., 2002
). Like
uvomorulin which, along with one of its key modulators, protein kinase C
(Winkel et al., 1990
;
O'Sullivan et al., 1993
), has
been implicated in blastomere polarization
(Fleming et al., 1994
), Ahnak
is under the control of protein kinase B/C
(Hashimoto et al., 1995
;
Sussman et al., 2001
), and may
thus localize to sites of cell-cell contact as outer cells epithelialize into
mesothelium.
A model of allantoic differentiation
On the basis of these and previous findings, we propose a model of
differentiation of allantoic mesoderm
(Fig. 12). All of the data
point to roles for intrinsic cues that may control allantoic cell age, as well
as roles for cell position, and signaling from the posterior primitive
streak.
|
Step 2. The distal allantoic region differentiates into angioblasts (Fig. 12B)
Twelve hours after the bud appears, FLK1-positive angioblasts differentiate
in the distal allantoic region, where mesoderm is oldest
(Downs and Harmann, 1997;
Kinder et al., 1999
)
(Fig. 12B, dark pink color).
Several hours later, distal angioblasts coalesce into identifiable endothelial
tubules (Downs et al., 1998
)
(Fig. 12E,F). The sequence of
angioblast differentiation and endothelial tube formation then proceeds down
the length of the allantois with striking regularity
(Fig. 12E,F)
(Downs et al., 1998
), strongly
suggesting that cell age plays a major role in this event. The importance of
cell age is further underscored by uniform cell proliferation along the
allantoic projection (Downs and Bertler,
2000
); constant cell turnover could ensure that differentiation of
each allantoic region takes place in turn. Moreover, whole explanted
headfold-stage allantoises invariably vascularized with distal-to-proximal
polarity according to a precise temporal program
(Downs et al., 2001
), and
proximal allantoic subregions, which are initially relatively negative for
FLK1 (Downs et al., 1998
),
also acquired abundant FLK1 in isolation
(Fig. 10J) (this study).
Thus, a series of downstream genetic events would be activated until a
final gene product triggers the expression of Flk1 in the distal
allantoic region, where mesoderm is oldest. Consequently, cell age, possibly
the result of an internal timing mechanism initiated by Hox gene
products in the bud (Fig.
12A), and cell position, ultimately coincide. An example of a
candidate regulatory Hox gene is Hoxb5, whose product has
been implicated in controlling expression of Flk-1
(Wu et al., 2003), although
its localization to the allantois has not yet been reported.
Step 3. Appearance of chorio-adhesive cells (Fig. 12C)
By 8.0 dpc, FLK1 is found in the distal two-thirds of the allantois
(Downs et al., 1998)
(Fig. 12E,F). Together with
cell proliferation and cavitation, sustained activity of the streak results in
lengthening of the allantois. Then, at a distance of approximately 220 µm
from the streak, VCAM1 becomes visible in distalmost mesothelial cells
(Fig. 12C, light green color).
Thus, chorio-adhesive cells have formed.
Results of this and previous studies suggest a model of chorio-adhesive
cell formation that, like endothelium, involves both cues intrinsic to the
allantois and an internal timing mechanism (reviewed by
Downs, 1998). In addition,
signaling from the primitive streak appears to be involved (Figs
10,
11). Saitou et al.
(Saitou et al., 2002
) have
suggested the presence of a localized signaling center within the posterior
streak. Factors emanating from the posterior streak could be extended to the
allantois, inhibiting expression of Vcam1 in proximal mesoderm. A
downstream target might be the homeobox-containing forkhead transcription
factor, Foxf1, as in Foxf1 mutants Vcam1's
expression domain is expanded to include the proximal allantoic region
(Mahlapuu et al., 2001
).
Step 4. The allantois contains a distal-to-proximal vascular network; distal regionalization of VCAM1 is maintained (Fig. 12D)
In this step, we propose that factors emanating from the streak continue to
suppress VCAM1 to a distance of 220 µm. Endothelium has spread all the way
down to the base of the allantois (Fig.
12D-F), at which time, the allantoic vasculature amalgamates with
those of the yolk sac and fetus (Downs et
al., 1998; not shown).
Chorio-adhesive cells
The VCAM1-negative proximal region may, in future, winnow out genes that
regulate chorio-adhesive cell formation. For example, Tbx4
nullizygous allantoises failed to grow far enough to fuse with the chorion and
did not exhibit VCAM1 (Naiche and
Papaioannou, 2003). In light of findings here, absence of VCAM1
may be due to the foreshortened mutant allantois' failure to escape the
streak's suppressive influence, rather than to defective genetic interaction
between Tbx4 and Vcam1.
Intriguingly, despite a conspicuous absence of VCAM1, 8-hour proximal
regions nonetheless often made contact with the chorion. Although the extent
to which non-VCAM1- and VCAM1-containing proximal allantoic regions penetrate
the chorion awaits further study, this observation is consistent with
transgenic knockouts. These revealed that 50% of Vcam1 nullizygous
embryos undergo chorio-allantoic union before dying at approximately 11.5 dpc
(Gurtner et al., 1995;
Kwee et al., 1995
). Potential
explanations for this intriguing observation have already been discussed
(Gurtner et al., 1995
;
Kwee et al., 1995
;
Downs, 2002
).
Why VCAM1 is normally absent from the proximal allantoic region is not
clear, but given that a major allantoic function is to unite with the chorion
and form the umbilical vasculature, repression might ensure that the proximal
region cannot fuse with the chorion. If it, like the distal region, spread
along the chorionic surface (Downs,
2002), compression rather than elongation of the umbilical cord
might occur. Although short cords are extremely rare, they are usually
associated with fetal malformations
(Benirschke, 1998
).
Conclusions and future perspectives
Finally, although three cell types have thus far been characterized in the
allantois, it is not known whether vascular smooth muscle cells arise from the
allantois itself or are contributed to the allantois from the chorion.
According to recent results (Yamashita et
al., 2000; Motoike et al.,
2003
), vascular smooth muscle cells may originate from
FLK1-positive precursor cells.
The function and origin of other cell types, known to exist only through
gene expression patterns, remain unclear or unknown. For example,
VCAM1-positive cells are closely associated with the FLK1-positive endothelium
in the distal and not the proximal core, but their significance in allantoic
development has not been elucidated (Downs
et al., 2001; Downs,
2002
). One possibility is that, once the chorio-allantoic fusion
surfaces break down, VCAM1-positive distal cells bridge the connection between
the FLK1-containing endothelial cells and the chorionic ectoderm
(Downs, 2002
), binding to
4-integrin present in the chorion
(Bowan and Hunt, 1999
). In
addition to core VCAM1,
4-integrin has been identified in a small cell
population in the proximal region of the allantois, which is continuous with
the amnion (Downs, 2002
).
These cells may be involved, through integrin-mediated cell motility, in
expanding the amnion during fetal growth.
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ACKNOWLEDGMENTS |
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