Download The Cellular Basis of Gastrulation in Xenopus laevis: Active

AMER. ZOOL., 24:589-603 (1984)
The Cellular Basis of Gastrulation in Xenopus laevis:
Active, Postinvolution Convergence and
Extension by Mediolateral Interdigitation
R. E. KELLER
Department of Zoology, University of California,
Berkeley, California 94720
SYNOPSIS. Time-lapse videomicrographic and SEM analyses of normal and microsurgically altered gastrulation show that the morphogenetic movements of the dorsal marginal
zone (DMZ)—extension, convergence, and involution—all result from behavior that occurs
after the marginal zone has involuted. Before its involution, the DMZ shows no detectable
capacity for autonomous convergence or extension. If its involution is prevented, the DMZ
will show convergence and extension but only at developmental stages at or beyond the
stage at which it normally would have involuted. Thus autonomous convergence and
extension, which have been ascribed to the DMZ are, in fact, properties of the dorsal
mesodermal mantle (DMM) and the archenteron roof. SEM analysis of cell shape and
packing patterns, suggest that cells of the DMM merge (interdigitate) mediolaterally,
between one another, beginning just beyond the point of involution. This behavior is
thought to reduce the width and increase the length (postinvolution convergence and
extension) of the DMM. The decrease in circumference (width) at the vegetal-most part
of the newly involuted DMM forms a constriction ring just inside the blastopore. Constriction and concurrent elongation of the DMM act in concert to move the blastoporal
lip vegetally. The DMZ is passively pulled vegetally and over the blastoporal lip as deep
cells are recruited for participation in mediolateral interdigitation at the vegetal end of
the DMM.
REVIEW OF GASTRULATION IN
XENOPUS LAEVIS
Gastrulation in the anuran amphibian,
Xenopus laevis, occurs by a rolling over the
blastoporal lip (involution) of the superficial layer of the marginal zone to form the
endodermal roof of the archenteron, and
the concurrent involution of the deep
region of the marginal zone to form the
mesodermal mantle (Keller, 1975, 1976).
Xenopus has no mesoderm on the surface
(see Nieuwkoop and Florschutz, 1950; Keller, 1975, 1976), in contrast to other
anurans and urodeles that have been studied (see Vogt, 1929; Pasteels, 1942). As the
marginal zone involutes, the animal region
undergoes an increase in area, or epiboly
(see Vogt, 1929; Keller, 1978). During
epiboly, the dorsal marginal zone (DMZ)
lengthens greatly (extension) and narrows
(convergence). Extension supplies more
1
From the Symposium on Gastrulation presented
at the Annual Meeting of the American Society of
Zoologists, 27-30 December 1982, at Louisville, Kenlucky.
589
material to the blastoporal lip than is involuted; thus the blastoporal lip moves vegetally across the yolk plug (see Keller, 1981).
Convergence of the DMZ results in concurrent constriction of the blastopore.
Because most convergence and extension
occur in the dorsal sector, the dorsal and
dorsolateral lips move much farther across
the yolk plug than the lateral and ventral
lips, and therefore the blastopore closes
eccentrically over the ventral region of the
yolk plug (see Keller, 1975).
Concurrent with the onset of involution
of the deep region of the DMZ, bottle cells
form by apical constriction and apical-basal
elongation of superficial, epithelial cells in
the dorsal region (see Holtfreter, 1943a,
b; Baker, 1965; Perry and Waddington,
1966). These changes in cell shape result
in formation of the blastoporal groove by
invagination—the bending of a cell sheet
(see Lewis, 1947). Further deepening of
the groove to form the archenteron results
from vegetal extension and involution of
the blastoporal lips rather than invagination (see Keller, 1981). The roof of the
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R. E. KELLER
archenteron is attached tightly to the deep
mesodermal cells. These mesodermal cells
appear to migrate toward the animal pole
(Nakatsuji, 1975, 1976; Keller and Schoenwolf, 1977) and carry the archenteron roof
with them (Keller, 1981). More accurately,
the involuted mesoderm pulls the preinvolution material vegetally, over itself.
In urodeles, bottle cells were thought to
migrate into the interior of the gastrula
and pull everything inside (Holtfreter,
1943a, b). However, there are several arguments against this mechanism operating in
anurans (see Keller, 1981) and urodeles
(Daniel and Yarwood, 1939). In Xenopus,
an anuran, bottle cell removal does not
prevent extension, convergence, or involution (Cooke, 1975; Keller, 1981).
Spreading of the superficial layer occurs
by increase in area, flattening, and division
of superficial cells (Keller, 1978, 1980).
Deep cells do not move out into the superficial layer as they do in the urodele (see
Holtfreter, 1943a). The deep region
spreads by interdigitation of several layers
of deep cells to form fewer layers of greater
area. This interdigitation may be an active,
force-producing process in epiboly or a
passive response to forces generated elsewhere (Keller, 1980). Active interdigitation could presumably result in autonomous spreading and perhaps extension of
the DMZ (Keller, 1980). Isolation experiments on other amphibians (Holtfreter,
1939; Townes and Holtfreter, 1955; Ikushima and Maruyama, 1971) and rearrangement experiments by Schechtman
(1942) and others (see Spemann, 1938),
suggest that the extension of the DMZ is
an active, autonomous process. But in all
these works it is not clear whether the
observed extension occurred during the
gastrula stages or represented extension of
the notochord during neurula stages.
Changes in cell morphology, arrangement, and probably behavior, occur at the
point of involution, particularly in the deep
region (Keller and Schoenwolf, 1977; Keller, 1981). Only the deep cells of the marginal zone have the capacity to complete
these changes and involute (Keller, 1981).
It is not clear what these changes are, or
how thev function in involution. There are
changes in the extracellular matrix (Kosher
andSearls, 1973;Johnson, 1977a-d, 1984;
also see Lee et al., 1982) and cell surface
charge (Schaeffer et al., 1973) that may be
associated with the involuting cells.
The task at hand is to identify a population or populations of cells and their corresponding behavior patterns that bring
about the movements of gastrulation. Previously, the cellular basis of epiboly was
modeled in terms of two alternative mechanisms—active or passive interdigitation
of deep cells (Keller, 1980). Other work
strongly suggested that the deep region of
the marginal zone is the critical element in
the process of involution (Keller, 1981).
The goal of the present work was to determine what region of the gastrula can
undergo autonomous convergence and
extension and what cell behavior is
involved.
Microsurgical alteration of the DMZ and
analysis of the resulting gastrulation by
time-lapse videomicroscopy (TLV) and
scanning electron microscopy (SEM),
strongly suggests that the force-generating
process that brings about all these movements—convergence, extension, and involution—does not occur in the preinvolution DMZ but in the postinvolution dorsal
mesodermal mantle (DMM), beginningjust
inside the point of involution. It is proposed that extension, convergence, constriction of the blastopore, and involution
form a complex of processes that all result
from a common postinvolution process—
active, mediolateral interdigitation of deep
mesodermal cells. The principal elements
of this notion of gastrulation and the key
experiments supporting them will be set
forth here. Further description, more
detailed analyses, and tests of the major
hypotheses of this model will follow in subsequent publications.
EVIDENCE THAT ACTIVE EXTENSION AND
CONVERGENCE ARE
POSTINVOLUTION PROCESSES
Does active, autonomous convergence
and extension occur? If so, when and where
does it occur? In the classical literature (see
Spemann, 1938), extension and convergence were associated with the "marginal
XENOPUS GASTRULATION
591
FIG. 1. Microsurgical manipulations of the early gastrula are shown diagrammatically in sagittal (a, b, d) and
vegetal (c, e) views. Dorsal marginal zone (DMZ) was grafted to the animal pole (a). Patches of animal pole
were grafted to the dorsolateral marginal zone (b, c). Patches of the dorsal marginal zone were rotated 90
degrees clockwise (d, e). The superficial layer is shaded heavily and the deeper layer is shaded lightly. MM,
mesodermal mantle: BC, bottle cells; B, blastocoel.
zone," which was defined as the region that
is marginal to and above the blastopore.
But with time the marginal zone involutes
to form the mesodermal mantle and
archenteron roof (Vogt, 1929; Keller,
1975, 1976). The lateness of the autonomous extension and convergence shown in
classical works (see Spemann, 1938;
Schechtman, 1942) suggests that these
processes normally occur after involution,
and are properties of the DMM or the
archenteron roof rather than the marginal
zone. The following experiments support
this contention.
does not show this behavior. When grafted
to this site in an early gastrula (Fig. la), it
shows little or no extension (Fig. 2a) until
after it reaches the midgastrula stage. Then
it begins to elongate, arch above the surface of the host, and form a notochord (Fig.
2b-e). Usually, the end that begins extension and arches above the surface of the
host is the former vegetal end. The important point is that this extension occurs at
developmental ages equivalent to the midgastrula and beyond, when normally the
vegetal end of this region would have involuted.
Grafts of DMZ to the animal pole
Bilateral blockage of involution
If extension of the preinvolution DMZ
is active and autonomous, it would be
expected to occur when the DMZ is grafted
to the animal pole, a region that ordinarily
Grafts of animal pole deep cells to the
marginal zone will block involution in the
region of the graft (see Keller, 1981). If
bilateral grafts of deep and superficial layer
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R. E. KELLER
FIC. 2. A graft of early gastrula (stage 10) DMZ to the animal pole retains its initial shape until the midgastrula
(stage 10.5) (a), at which time it begins to extend at its vegetal end through the late gastrula (stage 11.5, b;
stage 12, c) and neurula (stage 15, d; stage 17, e).
FIG. 3. Grafts of patches of early gastrula animal pole (shaded) to the dorsolateral marginal zone block
involution on both sides of the DMZ. The DMZ extends beyond the blocking patches and begins to narrow
from the midgastrula stage (a) onward through the late gastrula (c). From the early neurula (d) through the
middle (e, f) and late neurula stages (g), the extension of the DMZ is dramatic. During the neurula stages,
the lateral and ventral lips of the blastopore expand and secondarily uncover the yolk plug (YP). This extension
of the DMZ (notochord) stretches the grafted patches (d-g).
XEMOPUS GASTRULATION
593
FIG. 4. Photographs of neurulae derived from early gastrula that received grafts of animal pole material to
the dorsolateral marginal zone. The grafted patches (pointers in a) can be seen on both sides of the DMZ
(notochord) which has extended between them. The DMZ invariably skews off to the right or left and extends
most of the distance across the newly exposed yolk plug. The yolk plug is re-exposed by extension (thick
arrows, b) of the lateral and ventral lips of the blastopore (thin arrows, b).
of the animal pole are grafted to the dorsolateral marginal zone (Fig. lb, c), the
DMZ between them is isolated in situ by
regions on either side that will not involute
(Fig. 3). The blastopore continues to constrict but the patches of animal material
refuse to move inside. The isolated dorsal
sector begins to narrow, arch above the
host, and extend beyond the lip of the blastopore (Fig. 3a-c). In the late gastrula (Fig.
3d) and neurula stages (Fig. 3e-g), the dorsal sector extends greatly. Histological sections (not shown) show these extensions to
consist of notochord. The extensions
stretch the grafted patches along their
flanks as they elongate (Fig. 4a, b). The
lateral and ventral lips begin to extend circumferentially in the late gastrula and neurula stages and dilate the blastopore (Fig.
3d-g; see arrows in Fig. 4b). Histological
sections of the resulting "ring embryo" (see
Schechtman, 1942) shows that the extending lateral and ventral sectors contain
somites. The secondary dilation of the blastopore and the associated extension of the
somitic mesoderm will be dealt with in
detail in a later publication. The important
point here is that the isolated, dorsal sector
constricts and extends from the midgastrula or late midgastrula stage onward.
594
R. E. KELLER
FIG. 5. A square patch of DMZ rotated 90 degrees clockwise in the early gastrula has begun to elongate to
the left at its vegetal end by the midgastrula stage (a). It fails to involute but continues to elongate around
the left side of the blastopore during the second half of gastrulation (b-c) and through the middle (d) and
late (e) neurula stages. Note the progressively greater extension at the former vegetal end (marker dots, ac). NP, neural plate; YP, yolk plug.
Ninety degree rotation of the DMZ
If a small patch of DMZ is rotated clockwise 90 degrees (Fig. Id, e), it fails to involute, and it fails to extend or spread significantly during early gastrulation. In the
midgastrula stage, it begins to extend (Fig.
5a), and by the late midgastrula stage, it
has extended in the proper direction, with
respect to its own axis, and around the left
side of the blastopore, but it fails to involute (Fig. 5b, c). Note that it is the former
vegetal end that begins to extend first (see
markers, Fig. 5). Extension continues into
the neurula stages and the redirected DMZ
forms notochord around the inside of the
enlarged left lip of the unclosed blastopore
(Figs. 5d, e, 6a). Rotation of the deep region
alone has similar effects (Fig. 6b), whereas
rotation of the superficial layer alone usually, but not always, has little effect (Fig.
6c).
The effect of 90 degree rotation is
dependent on the size of the rotated patch.
Small patches have less effect than large
patches. If a large patch is rotated clockwise 90 degrees, it is not deflected to the
left around the blastopore but behaves
independently. It elongates parallel to its
own animal-vegetal axis but perpendicular
to that of the host (Fig. 6d). The former
vegetal end (to the left, Fig. 6d) extends
above the surface of the host and narrows
in the process, whereas the opposite end
remains broad (pointers, Fig. 6d). The
extending DMZ often projects far from the
XENOPUS GASTRULATION
595
Fie. 6. A midneurula (stage 15) after 90 degree clockwise rotation of the DMZ (a) shows incomplete blastopore
closure and truncated neural plate (NP). The rotated DMZ has extended into the thickened, left lateral lip.
Removal and replacement without rotation has little effect (left in b). Rotation of the deep region alone has
nearly the effect of rotating both layers (right in b), whereas rotating the superficial layer alone usually has
little effect (c). A large patch of DMZ rotated clockwise 90 degrees narrows and extends to the left (arrow),
from its former vegetal end whereas the opposite end remains spread (pointers) by the late gastrula stage (g).
Magnifications: a, 52; b, 21; c, 37; d, 62.
surface of the embryo and sections show it
to consist of notochord (data not shown).
Three important facts emerge from these
experiments. First, the redirected DMZ
shows extension and convergence but
again, only at midgastrula stages and
beyond. Secondly, it shows extension and
convergence from the vegetal end first.
596
R. E. KELLER
Lastly, the DMZ can not involute when
presented to the blastopore at right angles
to its normal orientation.
Conclusions
lution (Keller, 1981). Grafts containing
animal pole deep cells will block involution. Animal pole deep cells will actually
pass through the zone of involution but
then stop on the other side where they
would be expected to leave the involution
zone and join the involuted material (see
Fig. 8b, Keller, 1981). This fact suggests
that involution, like extension and convergence of the outer (preinvolution) DMZ, is
a passive process, dependent on postinvolution events.
Firstly, the preinvolution DMZ shows no
detectable capacity to extend at developmental ages prior to the time at which it
normally would have involuted. If a capacity to extend autonomously exists prior to
the normal time of involution, it is too weak
to distort the surrounding, uncooperative
tissues in these experiments. This is
ANALYSIS OF POSTINVOLUTION EVENTS
unlikely. DMZs isolated in culture, without
adjacent tissues to resist their extension,
What postinvolution events drive the
appear to show little extension until at least convergence and extension of the dorsal
the late midgastrula stage (Holtfreter, sector of the gastrula? Do they occur in the
1939; Schechtman, 1942; Townes and deep region (the DMM) or the superficial
Holtfreter, 1955; Ikushima and Maru- endodermal epithelium (the archenteron
yama, 1971). The fact that the preinvolu- roof)? The answers are not clear. Grafting
tion DMZ will not extend autonomously experiments show that the processes of
until a developmental age when it normally convergence, extension, and involution all
would have involuted suggests that the appear to be less sensitive to perturbations
extension of the dorsal side of the embryo in the superficial layer than in the deep
is normally due to a property of the DMZ region (Keller, 1981). Thus, for the
after it has involuted to form the DMM moment, I will focus on the morphologies
and the archenteron roof.
and packing-patterns of the deep mesoSecondly, involution is not required for dermal cells that comprise the DMM as they
extension, since the DMZ will extend on relate to postinvolution convergence and
the outside of the embryo without extension. Earlier work focused on the fact
undergoing the process of bending over that as they leave the involution zone, the
the lip, either when grafted to the surface DMM cells appear to form a "stream" of
of the host, as was the case in the present cells in which individuals migrate toward
experiments, or in organ culture (see Holt- the animal pole on the inner surface of the
freter, 1939, 19436; Schechtman, 1942; preinvolution material (Nakatsuji, 1975,
Townes and Holtfreter, 1955). Thirdly, 1976; Keller and Schoenwolf, 1977). This
extension occurs first and most extensively work emphasizes the interaction of deep
from the vegetal end of explanted or redi- mesodermal cells with the inner surface of
the gastrular wall. I will return to this idea
rected patches of DMZ.
Fourthly, it is important to note that the and suggest that it be modified somewhat.
lateral side of a patch of DMZ rotated 90 Now I wish to examine the possibility that
degrees will not involute, even though it there are interactions between these
lies within the material of the marginal zone mesodermal cells that bring about postincapable of involution (see Keller, 1976, volution convergence and extension.
1981). This suggests that the material of
the marginal zone must be presented to the Morphology and pattern of DMM cells
involution zone with the proper axial oriThe overlying preinvolution material was
entation in order for involution to occur. dissected off, after fixation, and the outer
Lastly, involution itself appears to be surface of the involuted DMM was examdependent on events that occur after cells ined with stereo SEM by methods similar
have passed through the involution zone. to those described in detail elsewhere (KelThe type of deep cell is critical for invo- ler and Schoenwolf, 1977). Patterns of cell
XENOPUS GASTRULATION
arrangement and shape that may be related
to the processes of dorsal convergence and
extension were seen.
At the midgastrula stage, cells in the
DMM appear to be more tightly packed
with smaller intercellular spaces than those
lateral to the DMM (Fig. 7a, b). High magnification (Fig. 7c) shows that cells in this
region have less relief and are more tightly
packed than those located laterally. Many
are elongated and aligned transversely to
the animal-vegetal axis (Fig. 7c). This pattern is more pronounced among cells just
inside the zone of involution, at the vegetal
extremity of the mesodermal mantle, where
most are elongated parallel to the circumference of the blastopore (Fig. 7d). This
band of elongate cells extends laterally into
the dorsolateral sectors of the posterior
mesodermal mantle and is contiguous with
the shield of compacted cells extending
anteriorly from it (Fig. 7a, b).
In late gastrulation, a similar but longer
and narrower area is found in the DMM,
extending anteriorly from the zone of
involution (Fig. 8a, b). Cells in this region
are distinguished from those on either side
by the presence of large, lamellar protrusions, a tighter packing pattern with fewer
intercellular spaces and a much more flattened morphology. These characteristics
are shared with the corresponding area of
the midgastrula, but their expression is
subtle at the midgastrula whereas it is more
extreme in the late gastrula. This region
is clearly the developing notochord at the
late gastrula stage, and it is set off distinctly
from the paraxial mesoderm (Fig. 8b; also
see Fig. 2, Youn et ai, 1980). In the early
neurula, the notochord consists of a more
compact, narrow array of cells clearly separated from the prospective somite mesoderm lateral to it (Fig. 8c).
There is good evidence that the area of
unique cell shape and arrangement in the
DMM of the mid (Fig. 7), and late (Fig. 8)
gastrula stages are homologous to the
notochord (Fig. 8c) in the neurula. These
regions in the gastrula and neurula map as
prospective notochord (Keller, 1976, and
unpublished data). The less-ordered, indistinct packing-pattern and morphology of
this region of the midgastrula develops
597
without discontinuity into the highly
ordered cell shape and arrangement characteristic of the notochord in the neurula.
Convergence and extension by
mediolateral interdigitation?
There is no direct evidence pertaining
to the behavior of the DMM cells described
above in SEMs. There is indirect evidence,
however, that these cells undergo mediolateral interdigitation and repacking to
form an array that is narrower and longer
(convergence and extension). The dorsal
convergence of the DMM in the gastrula
and early neurula stages is extraordinary,
and this dramatic movement dorsally is
expressed primarily as an increase in length
(Keller, 1975, 1976), rather than as an
increased thickness, though the developing notochord does thicken slightly (Fig.
8b). These facts imply that DMM cells moving dorsally must repack, mediolaterally,
to form a mass of greater anterioposterior
extent. Direct evidence on this point is
needed.
The emergence of the dorsal packingpattern is spatially and temporally coincident with the increasing capacity for
autonomous extension in the second half
of gastrulation and in the neurula. The
packing-pattern and the capacity to extend
autonomously are both associated with the
prospective or developing notochord.
Notochord is invariably found in the protrusions that break away from the surface
of the gastrula (see Schechtman, 1942).
Thus a major part of the dorsal convergence and extension in the gastrula stage
and notochordal extension in the neurula
stages may be based on similar cellular
morphologies, packing-patterns and
behavior.
Though the capacity for convergence
and extension in the dorsal sector is normally coincident with the notochord anlage, such capacity is not strictly associated
with notochord formation. The lateral
blastoporal lips of "ring embryos"
described above and in Schechtman (1942)
contain only somites but participate in constriction of the blastopore and then
undergo dramatic extension. Moreover,
irradiated embryos that never form a
B
FIG. 7. A posteriodorsal view of the involuted mesodermal mantle of a midgastrula (stage 10.5) shows the DMM cells (pointers) characterized by
low relief, closer packing, and smaller intercellular spaces (a, b). High magnification clearly shows the difference between medial and lateral cells in
these respects (c). The vegetal end of the DMM, just inside the mesodermal involution zone (MIZ), shows closely packed mesodermal cells elongated
— —
1 AA.
U
OAA.
„
XENOPUS GASTRULATION
599
Fie. 8. A posteriodorsal view of the DMM of a late gastrula (stage 11.5) shows an elongate region of closepacked cells extending anteriorly from the dorsal mesodermal involution zone (MIZ) (a). Higher magnification
shows these cells, which are prospective notochord, to be more flattened, closely-packed, and bearing more
lamelliform protrusions than the somite mesoderm to either side (b). A dorsal view of the late gastrula (stage
13) shows the contrast between the cells of the notochord (N) and paraxial mesoderm (PM) (c). Magnifications:
a, 100; b, 250; c, 500.
proper notochord show extension (Malacinski and Youn, 1981).
CELLULAR MECHANISMS OF
GASTRULATIONS
Previous proposals
Previously, two alternative mechanisms
were proposed to explain spreading or
epiboly of the preinvolution region of the
gastrula (Keller, 1980, 1981). According
to the first, autonomous spreading of the
DMZ occurs by active, radial interdigitation of several layers of deep cells to form
fewer layers of greater area (see Fig. 19,
Keller, 1980). This proposal was supported
by the evidence from the literature, which
appeared to favor the active, autonomous
extension of the marginal zone and animal
region (see Keller, 1980). However, the
tests of these alternatives, presented here,
strongly suggest that spreading of the DMZ
is passive, and thus favor the second mechanism. According to this mechanism, the
superficial epithelium would be stretched
by tension generated at the blastopore, and
deep cells would rearrange and occupy the
additional space made available on the
inner surface of the superficial layer (see
Fig. 19e,f, Keller, 1980). The movement
of the deep cells would not, in itself, force
the spreading of the marginal zone, though
this is the only sense in which their behavior would be considered passive. Their
apposition to the inner surface of the
superficial epithelium and their repacking
to form fewer layers could be viewed as a
rachet mechanism—not actively forcing
the expansion of the epithelium but stabilizing it once it was stretched by external
forces.
A new mechanism: Active, postinvolution
convergence and extension by
mediolateral interdigitation
The above evidence suggests that the
external force that stretches the DMZ is
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R. E. KELLER
DMZ
FIG. 9. The Postinvolution Convergence and Extension Model is illustrated diagrammatically in a lateral
view of a midgastrula with the right dorsal and lateral marginal zone removed (a) to expose the dorsal
mesodermal mantle (DMM), and in a gastrula section midsagittally (b). The cells of the DMM (outlined) merge
dorsally by mediolateral interdigitation (inset, a), beginning just inside the zone of involution. The DMM
narrows (converges) as indicated by the arrows pointing dorsally in the DMM (a). Such narrowing is expressed
by lengthening (extension) of the DMM at the middorsal region (dashed arrow, a) and by formation of a
constriction zone in the DMM just inside the blastoporal lip. The constriction zone and the extension of the
DMM result in the DMM pushing vegetally, on the inside of the blastoporal lip (short arrows, a) and concurrently constricting the blastopore. As the DMM pushes vegetally (short arrow, b) on the inner surface of
the superficial epithelium (SE), the overlying dorsal marginal zone (DMZ) is towed vegetally (curved arrows,
b). Deep cells of the marginal zone (shaded) are recruited to join the posterior DMM (b). The bottle cells
(BC) anchor the superficial epithelium to the mesodermal mantel (see Keller, 1981).
most likely generated by postinvolution
events in the DMM. The following cellular
model of gastrulation, based on the arguments above, is proposed. The object of
proposing this model is to stimulate
research that will either support or refute
its principal elements, which are as follows.
Firstly, there are deep mesodermal cells
in the DMZ, spread far laterally on both
sides of the dorsal midline, that have, as
part of their morphogenetic repertoire, the
capacity to converge dorsally by active,
mediolateral interdigitation (Fig. 9a). Secondly, these cells begin to exercise this part
of their morphogenetic repertoire just after
they leave the zone of involution and join
the vegetal end of the mesodermal mantle
(Fig. 9a, b). There they merge between one
another and converge on the dorsal midline. This behavior is characteristic of and
continues to occur among involuted cells
along the length of the DMM as well as at
its vegetal end. Thus width of the vegetal
end of the DMM decreases and forms a
constriction ring in the mesodermal mantle just inside the blastoporal lip (Fig. 9a,
b). Because this constriction ring acts below
the equator of a spherical embryo, it tends
to pull the blastoporal lip vegetally, across
the yolk plug (Fig. 9a). Because convergence begins earlier in dorsal and dorsolateral sectors, the blastoporal lips in these
regions move greater distances across the
yolk plug and close the blastopore eccentrically, which is the case (Keller, 1975). In
addition, the mediolateral interdigitation
XEXOPUS GASTRULATION
of cells toward the dorsal midline would
narrow the DMM along its entire length
(convergence) and increase its length, causing extension vegetally across the yolk plug
in concert with the constriction movement
of the blastoporal lips.
Lastly, preinvolution (marginal zone)
deep cells are continually recruited to join
the vegetal edge of the DMM. The requirement for successfully doing so is the adoption of the behavioral rules (or rule) that
underlie active, mediolateral interdigitation. Determining the cellular, motile
behavior underlying mediolateral interdigitation of DMM cells is essential in our
understanding of gastrulation in Xenopus
and probably many other anurans as well.
Holtfreter (19436) and Townes and
Holtfreter (1955) noted that the isolated
DMZ would elongate and form notochord
if left intact but would form notochord tissue without elongating if dissociated and
reaggregated. This fact might suggest that
the order of neighbor relationships are
required expression of a morphogenetic
function (extension) but no histodifferentiation (of notochord tissue). Alternatively,
it could reflect a greater sensitivity of the
former process to the trauma of dissociation.
601
involves lateral and ventral sectors. Thus
these processes begin earlier, continue
longer, and involve more cells in the dorsal
sector than in lateral and ventral sectors
(see Keller, 1976). However, Schechtman
(1942) suggested that lateral and ventral
sectors have properties that are different
from the dorsal sector and that these
regions must interact in order to produce
gastrulation. His results and interpretation
are consistent with those presented here in
that the local rule of cellular behavior—
the postinvolution, mediolateral interdigitation—must operate with the dorsal, lateral and ventral sectors' circumblastoporal
continuity in order to be effective in producing involution. However, it is not clear
whether differences in dorsal and lateral
or ventral behavior are matters of timing,
degree, or character of behavior. It is also
not clear now convergence and extension
of the marginal zone at a given stage is
related to the notochordal and somitic
mesoderm. It is clear that anterioposteriorly organized events—extension and
involution—are intimately coupled to dorsoventrally organized events—dorsal convergence and constriction of the blastopore—because they are results of a
common process—postinvolution mediolateral interdigitation.
Mediolateral and anterioposterior
organization of convergence
and extension
Postinvolution convergence and
transitions in cell behavior
The above mechanism does not apply to
the initial movement of the blastoporal lip
vegetally and the initial involution of the
marginal zone. The anterior (early involuting) mesodermal mantle has little capacity for convergence and extension, based
on the evidence presented here and by
Schechtman (1942) and, in fact, does not
converge or extend (Keller, 1976). Studies
in progress show that other processes,
including bottle cell formation and mesodermal migration, have a greater role in
the initial movements; these will be
described elsewhere.
The degree of convergence and extension increases progressively from anterior
to posterior in the mesodermal mantle.
Moreover, it begins dorsally at about the
midgastrula stage, and progressively
The postinvolution convergence and
extension model is economical in terms of
the cellular behavior required since it
accounts for the movements of convergence, extension, and involution with one
basic cellular, motile process—active
mediolateral interdigitation—acting in a
specific set of global geometrical constraints. One transition in cellular behavior
is required just after the cells involute. In
contrast, the idea that the preinvolution
marginal zone actively extends toward the
blastopore requires that the deep cells of
the marginal zone first interdigitate radially and the entire marginal zone would
have to form a stiff plate, capable of pushing toward the involution zone (see Keller,
1980). Then, in the involution zone, the
cells must change behavior again, in such
602
R. E. KELLER
embryonalen Formbildung. Arch. Exp. Zella way as to allow the stiff plate to bend
forsch. besonders Gewebequecht 23:169-209.
around the blastoporal lip. Finally, on leavHoltfreter, J. 1943a. Properties and function of the
ing the involution zone, they would have
surface coat in amphibian embryos. J. Exp. Zool.
to change behavior again and migrate on
93:251-323.
the roof of the preinvolution material, Holtfreter, J. 1943A. A study of the mechanics of
gastrulation. Part I. J. Exp. Zool. 94:261-318.
toward the animal pole (see Nakatsuji,
Ikushima,
N. and S. Maruyama. 1971. Structureand
1975, 1976; Keller and Schoenwolf, 1977).
developmental tendency of the dorsal marginal
Such mechanical and behavioral complexzone in the early amphibian gastrula. J. Embryol.
ity seems unlikely.
Exp. Morphol. 25:263-276.
Johnson, K. 1977a. Changes in the cell coat at the
onset of gastrulation in Xenopus laevis embryos.
J. Exp. Zool. 199:137-143.
Johnson, K. 19776. Extracellular matrix synthesis in
Earlier work suggested that the invoblastula and gastrula stages of normal and hybrid
frog embryos. I. Toluidine blue and lanthanum
luted deep mesodermal cells were orgastaining. J. Cell Sci. 25:313-322.
nized as a stream of cells that actively
K. 1977c. Extracellular matrix synthesis in
migrate upwards, as individuals, toward the Johnson,
blastula and gastrula stages of normal and hybrid
animal pole, on the inner surface of the
frog embryos. II. Autoradiographic observations
preinvolution material (see Nakatsuji,
on the sites of synthesis and mode of transport
of galactose- and glacosamine-labelled materials.
1975, 1976; Keller and Schoenwolf, 1977).
J. Cell Sci. 25:323-334.
This is probably true of the early involutK. 1977a1. Extracellular matrix synthesis in
ing regions when extension and conver- Johnson,
blastula and gastrula stages of normal and hybrid
gence have no major role, but in the late
frog embryos. III. Characterization of galactoseinvoluting regions (posterior mesodermal
and glucosamine-labelled materials. J. Cell Sci.
25:335-356.
mantle) the coordinated interactions
between mesodermal cells that underlie Johnson, K. E. 1984. Glycoconjugate synthesis during gastrulation in Xenopus laevis. Amer. Zool. 24:
mediolateral interdigitation are probably
605-614.
more important, at least in Xenopus. Such Keller, R. 1975. Vital dye mapping of the gastrula
may not be true of the urodeles, such as
and neurula of Xenopus laevis. I. Prospective areas
and morphogenetic movements of the superficial
the Mexican axolotl, which appear to have
layer. Develop. Biol. 42:222-241.
a less cohesive mesodermal mantle in which
R. 1976. Vital dye mapping of the gastrula
cells migrate as individuals (see Nakatsuji, Keller,
and neurula of Xenopus laevis. II. Prospective areas
1984).
and morphogenetic movements of the deep
region. Develop. Biol. 51:118-137.
Keller, R. 1978. Time-lapse cinemicrographic analACKNOWLEDGMENTS
ysis of superficial cell behavior during and prior
to gastrulation in Xenopus laevis. J. Morph. 157:
I wish to thank Steven D. Black, Robert
223-248.
Gimlich, and George Oster for their con- Keller,
R. 1980. The cellular basis of epiboly: An
tributions in stimulating discussions on gasSEM study of deep cell rearrangement during
trulation and Paul Tibbitts and John Shih
gastrulation in Xenopus laevis. J. Embryol. Exp.
Morphol. 60:201-234.
for their technical assistance. This work
R. 1981. An experimental analysis of the role
was supported by NSF Grant PCM81- Keller,
of the bottle cells and the deep marginal zone in
10985 to the author.
gastrulation of Xenopus laei'is. J. Exp. Zool. 216:
81-101.
Keller, R. and G. Schoenwolf. 1977. An SEM study
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