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35:358-371 (1995)
Target Recognition by Mesenchyme Cells During Sea Urchin Gastrulation l
JEFF
HARDIN
Department ofZoology, University of Wisconsin, 1117 W. lohnson St., Madison, Wisconsin 53706 SYNOPSIS. During sea urchin gastrulation, mesenchyme cells must migrate
to the correct locations and attach to specific sites within the embryo.
Primary mesenchyme cells (PMCs) aggregate into two major clusters in
the ventrolateral region of the embryo and other PMCs form an elaborate
pattern that presages the skeletal pattern of the larva. Numerous exper­
iments, including alteration of dorsoventral differentiation of the ecto­
derm with NiCl z, indicate that the ectoderm provides much of the pattern
information to which these cells respond. Secondary mesenchyme cells
(SMCs) attach to the ectoderm at a specific site near the animal pole at
the end ofgastrulation, undergoing a dramatic change in protrusive behav­
ior as they do so. Experiments indicate that the target region to which
they respond is restricted to a small domain that will form the larval
mouth, and that this region is unique in its ability to elicit these changes
in behavior. A third population of patterned mesenchyme has recently
been identified by their expression of the sea urchin homologue of the
Drosophila transcription factor, snail. Snail expressing cells form two
bilateral clusters near the sites where the larval arms will form as gastru­
lation ends. Finally, we have identified two interactions between cells in
the early embryo that seem to be important for the establishment of
pattern information for mesenchyme: (1) interactions between macromere
descendants and neighboring cells can establish new bilateral patterning
sites for PMCs, and (2) interactions betw~en the animal pole and cells in
a more vegetal location appear to result in induction of the oral field and
associated pattern information for both PMCs and SMCs.
in reliable locations. One of the great out­
standing questions in the study of gastru­
lation is how mesenchymal cells within the
gastrula engage in such coordinated mor­
phogenetic movements as the incipient body
plan emerges, and how the response of such
cells to pattern information in the embryo
modulates these behaviors. Several model
systems have been used to address this
question. Well studied examples of the con­
trol of mesenchymal migration during gas­
trulation include the deep cells that form
the embryo proper in teleosts (Wourms et
al., 1972; Trinkaus et aI., 1991; Ho, 1992),
the ingression ofcells from the presumptive
epiblast to form the posterior margin of the
developing avian embryo (Stern, 1990,
From the Symposium The Role ofCell-Cell lnter­ 1991) and the migration of head mesoderm
actions and Environmental Stimuli in the Development
ofMarine Invertebrates presented at the Annual Meet­ cells in the amphibian embryo (Winklbauer
et al., 1991; Keller and Winklbauer, 1992;
ing of the American Society of Zoologists, 27-30
December 1993. Los Angeles, California.
Boucaut et al., 1991). Another model sys­
358 INTRODUCTION
The movements ofgastrulation are a par­
ticularly dramatic example of the response
of tissue sheets and freely migrating mes­
enchymal cells to pattern information within
the embryo. As tissue sheets involute,
invaginate or spread during gastrulation,
they do so based on precise partitioning of
tissue territories in which different morpho­
genetic programs are carried out. The same
is true of mesenchymal cells; different pop­
ulations of mesenchymal cells respond very
differently to pattern information within the
embryo, resulting in the migration and
aggregation ofvarious types ofmesenchyme
I
TARGET RECOGNITION BY MESENCHYME DURlNG SEA URCHIN GASTRULATION
359
ventral +---"""'i~~ dorsal (aboral)
(oral)
animal
secondary
mesenchyme
apical plate
archenteron
vegetal
view from
oral side
spicule
rudiment
lateral view
late gastrula/prism
FIG. I. A schematic representation of pallerning sites for mesenchyme in the sea urchin late gastrula, viewed
from the the ventral side (left) and the left side (right). For description of labeled structures, see the tex\.
invagination), the archenteron extends lf4­
'12 of the way across the blastocoel. A short
pause follows primary invagination, after
which the archenteron resumes its elonga­
tion (secondary invagination). At about the
time secondary invagination begins, cells at
the tip of the archenteron (secondary mes­
enchyme cells) become protrusive, extend­
ing long filopodia into the blastocoel. Even­
tually the archenteron elongates across the
blastocoel, and its tip attaches to the ventral
ectoderm near the animal pole (Fig. 1). As
the archenteron invaginates, primary mes­
enchyme cells migrate and eventually local­
ize in a precise pattern. Two major bilateral
clusters ofPMCs appear in the ventrolateral
region of the embryo, near two bilateral
thickenings in the ectoderm, so that the
embryo has a morphologically obvious dor­
soventral axis (Fig. 1). Other PMCs form a
OVERVIEW OF GASTRULATION IN THE ring in the vegetal half of the embryo, which
SEA URCHIN EMBRYO connects with the ventrolateral clusters in a
Just prior to gastrulation in the sea urchin characteristic fashion (see Ettensohn, 1990
embryo, the epithelium at the vegetal pole for an in-depth discussion of skeletal pat­
of the blastula flattens and thickens to form terning). The PMCs eventually fuse with one
the vegetal plate. Following the flattening of another to form a syncytium, and they
the vegetal plate, primary mesenchyme cells secrete the skeletal rods, or spicules, of the
ingress into the blastocoel. Then the vegetal larva (for reviews of skeleton formation in
plate begins to bend inward to form a short, the sea urchin larva see Decker and Len­
squat cylinder, the archenteron. During this narz, 1988; Benson and Wilt, 1992; Etten­
initial phase of invagination (primary sohn and Ingersoll, 1992). lntimately the
tern in which the patterning and morpho­
genesis of mesenchyme has been studied is
the sea urchin embryo. Because of its simple
organization, the ability to observe mor­
phogenetic movements directly in living
embryos, and because of its rich historical
context, the sea urchin embryo is a good
system in which to study ways in which
axial patterning influences the behavior of
mesenchyme at the single-cell level (for
reviews, see Wilt, 1987; Ettensohn, 1991;
Hardin, 1990, 1994a, b). In the following
sections, classical and new experimental
evidence for regionally specific patterning
of the ectoderm of the sea urchin embryo
will be presented. In addition, experiments
that indicate that cell interactions are
required for the emergence of this pattern
information will be discussed.
360
JEFF HARDIN
tip of the archenteron fuses with the ventral
ectoderm near the animal pole to form the
larval mouth. As the pluteus larva develops,
the archenteron becomes tripartite, and
undergoes regional differentiation to form
an esophagus, stomach, and intestine.
EcrODERMAL CuEs FOR LoCALIZATION OF PRIMARY MESENCHYME CELLS After their ingression into the blastocoel,
primary mesenchyme cells (PMCs) remain
at the vegetal plate briefly before migrating
within the interior of the embryo. The basic
mechanisms of migration of these cells have
been extensively reviewed elsewhere (Gus­
tafson and Wolpert, 1963, 1967; Solursh,
1986; Ettensohn and Ingersoll, 1992; Har­
din, 1990, 1994a, b). It is clear from a num­
ber of studies that PMCs respond to pattern
information present within the ectoderm
during gastrulation. The precise ring pattern
formed by these cells, the aggregation of
PMCs into two ventrolateral clusters, and
the elaboration of details of skeletal pattern
are all influenced by ectodermal cues. One
of the earliest cues for PMC localization
appears to reside in the vegetal plate. Fol­
lowing their ingression, PMCs remain at the
vegetal pole of the embryo for a noticeable
period of time prior to their migration away
from the vegetal plate. If PMCs are dis­
placed to the animal pole by centrifugation
(Okazaki et al., 1962) or transplanted into
the animal pole (Ettensohn and McClay,
1986), or when PMCs arise from single
micromeres incorporated ectopically (Wray
and McClay, 1988), they migrate to the veg­
etal plate region and remain there until
undisturbed PMCs migrate away from the
vegetal plate (Ettensohn and McClay, 1986).
Subsequent aspects ofPMC patterning also
appear to be conditioned by interaction of
PMCs with the ectoderm. Okazaki and col­
leagues noticed that the ventrolateral clus­
ters form atop or very near regions of thick­
ened ectoderm that produce an optical effect
reminiscent of a Japanese fan (Okazaki et
aI., 1962). When these thickenings are dis­
placed by vegetalizing with lithium chlo­
ride, the ring and clusters also shift (Wolpert
and Gustafson [1961]; see the article by Wilt
et al. [1995] for further information on the
effects of LiCI on sea urchin develop­
ment). Gustafson and Wolpert (1963 , 1967)
suggested that the interaction ofPMCs with
the ventrolateral thickenings was largely due
to physical considerations, where presumed
adhesive sites would be found at a higher
density.
Other distributed guidance cues for PMCs
within the em bryo may exist, based on stud­
ies of pattern regulation. The classic studies
of Driesch (1900) and careful studies by
Takahashi et al. (1979) indicate that in 112
and 1/4 dwarf larvae, the skeleton is capable
of size regulation. Even more dramatic evi­
dence for such regulative control was pro­
vided by Ettensohn. When 2-3 times the
normal number of PMCs are transplanted
into a host embryo, the resulting skeletal
pattern is nonetheless normal (Ettensohn,
1990).
To provide a direct test of the extent to
which the ectoderm influences patterning of
PMCs, we have used radialized embryos as
a tool for studying patterning of primary
mesenchyme cells. In particular, we have
used nickel chloride treatment to produce
embryos in which all traces of bilateral sym­
metry are abolished (Hardin et aI., 1992).
Based on in situ hybridization and immu­
nostaining, one of the primary effects of
nickel seems to be to alter the distribution
of ectodermal tissues in the embryo, result­
ing in an overproduction of ventral (oral)
ectoderm and a corresponding decrease in
the amoun t of dorsal (aboral) ectoderm that
is produced (Hardin et aI. , 1992). The cil­
iated band, which normally forms at the
boundary between dorsal and ventral ecto­
derm, is concomitantly shifted to a vegetal
position. Embryos radialized in this fashion
using nickel chloride are a useful tool for
studying ectodermal cues for PMC pattern­
ing for several reasons. Since the ventrolat­
eral clusters normally form as two foci near
the vegetal pole close to the position of the
ciliated band, the position of spiculogenic
clusters should be shifted in nickel treated
embryos concomitant with the new posi­
tions ofectodermal territories in the embryo,
if these anatomical markers are reliable pre­
dictors of sites of pattern formation. In
addition, it is possible to perform reciprocal
transplants ofPMCs between nickel-treated
and normal embryos to test whether or not
TARGET RECOGNITION BY MESENCHYME DURING SEA URCHIN GASTRULATION 361 A
..
~
;
,J
,
,
4
>:~' ..
"
"\0.
.-.
\
~
FIG, 2. Effects of Ni0 2 on skeletal pattern in Lytechinus variegatus visualized with polarization microscopy
(adapted from Hardin et al,. 1992, with permission) (a) Prism stage embryo radialized with 0.5 mM NiCI 2 • (b)
Normal pluteus. Bar = 20 /Lm.
the ectoderm is a primary source of pattern
information for spiculogenesis.
Skeletal pattern is severely disrupted in
nickel-treated embryos. Rather than form­
ing the normal two bilateral triradiate skel­
etal elements, nickel-treated embryos pro­
duce as many as a dozen small triradiate
elements, disposed in a radial pattern near
the vegetal margin of the embryo (Fig. 2).
As these skeletal elements grow, they result
in an embryo with severe disruption ofskel­
etal pattern, although the total number of
PMCs and the amount of skeletal material
produced appears to be normal (Hardin et
al.) 1992). To determine whether the pri­
mary defect in nickel-treated embryos
resides with their PMCs or with the ecto­
dermal environment, reciprocal transplants
were performed between nickel-treated and
normal embryos (Armstrong et al., 1993).
When a normal complement ofPMCs from
nickel-treated embryos is transplanted into
normal host embryos from which all PMCs
have been previously removed, the result­
ing skeletal pattern is completely normal.
Conversely, when PMCs are removed from
nickel-treated embryos and a full comple­
ment of normal PMCs is subsequently
added, the skeletal pattern is completely
radialized. These results indicate that the
source of the ectoderm largely determines
the pattern that the PMCs adopt, at least
regarding bilaterality. In addition, by
repeating the experiments of Ettensohn
(1990) using nickel-treated rather than nor­
mal embryos, it can be shown that skeletal
size and pattern are still regulated in radi­
alized embryos, despite obvious disruptions
in skeletal morphology (Armstrong et al.,
1993). As discussed below, other regionally
specific aspects of skeletal pattern are also
under ectodermal control. However, finer
aspects of skeletal pattern, e.g., the intricate
fenestrations that are elaborated along the
length of various skeletal rods, appear to be
an endogenous characteristic of the species
from which the PMCs are derived, based
on heterospecific transplants (Armstrong
and McClay, 1994).
EcrODERMAL CuEs FOR LocALIZATION OF
SECONDARY MESENCHYME CELLS
Time-lapse cinemicrographic studies by
Gustafson and coworkers thoroughly doc­
umented the basic protrusive behavior of
SMCs in Psammechinus miliaris (reviewed
by Gustafson and Wolpert, 1963, 1967).
These studies suggested that the filopodia
of secondary mesenchyme cells "ran­
domly" explore the blastocoel, undergoing
continual cycles of extension, attempted
attachment, and retraction. As gastrulation
362
JEFF HARDIN
ends SMCs undergo a change in their behav­
ior; much of their protrusive activity sub­
sides, and they may become more loosely
associated with the tip of the archenteron
(Dan and Inaba, 1968; Gustafson and Kin­
nander, 1960). Eventually, several hours
after gastrulation is over, the tip of the
archenteron attaches to the stomodeum (an
invagination of the oral ectoderm) and fuses
with it to form the mouth of the pluteus
larva (Gustafson and Kinnander, 1960).
Attachments ofSMCs during gastrulation
Given the apparently random explora­
tory behavior ofSMCs, how do they reliably
attach the tip of the archenteron to the ani­
mal pole region as gastrulation ends? Classic
observations raised several possibilities.
Gustafson suggested physical cues were
largely responsible, including (l) differences
in surface topography at contact sites
between epithelial cells (Gustafson, 1963),
(2) tissue proximity (Gustafson and Wol­
pert, 1967; Gustafson, 1969), and (3) tissue
curvature, which might allow access of fil­
opodia to non-specific adhesive sites in some
regions but not others (Gustafson, 1963;
reviewed by Gustafson and Wolpert, 1967).
In addition, Gustafson suggested that the
ventral ectoderm might be generally more
adhesive than the dorsal ectoderm, which
would result in localization of both primary
mesenchyme cells and later, secondary mes­
enchyme cells (Gustafson, 1969). Other
observations, however, suggested that there
might be more specific guidance cues resi­
dent near the animal pole. In several Jap­
anese species (Dan and Inaba, 1968) and in
Lytechinus variegatus (reviewed in Trin­
kaus, 1984) filopodia initially extend lat­
erally, and only late in gastrulation do they
extend upward. This suggested that although
the essential motile program of SMCs
involves continual cycles of random filo­
podial extension, attachment and eventual
withdrawal, there might be unique pattern
information to which they respond near the
animal pole.
A n animal pole "target" exists for SMCs
We reexamined the attachment ofSMCs
to the animal pole region in some detail
(Hardin and McClay, 1990). At the end of
gastrulation the tip of the archenteron makes
contact with the ectoderm near a thickened
region of epithelium, the apical plate (cf
Fig. 1). At this time the exploratory behav­
ior of the filopodia largely ceases, marking
the end of gastrulation. Based on real-time
analysis of the residence times of attached
filopodia, protrusions that make contact
with the ectoderm in the apical plate region
remain attached 20-50 times longer than
attachments observed at any other site along
the blastocoel wall (Hardin and McClay,
1990). The SMCs bearing the long-lived fil­
opodia eventually change their behavior as
they flatten and spread onto this region. In
some species, such as Lytechinus variegatus,
this region lies near the animal pole; in other
species, such as Strongylocentrotus purpur­
atus, it is located on the ventral side of the
animal hemisphere (Hardin and McClay,
1990).
The normal behavior of SMCs suggests
that the animal pole region serves as a "tar­
get" for filopodial attachment, and several
experiments indicate that SMCs do in fact
respond uniquely to this region. First, when
the animal pole region is pushed toward the
tip of the archenteron with a micropipette
(Fig. 3) or when embryos are trapped in
Nitex mesh of the appropriate dimensions,
SMCs interact with the animal pole pre­
cociously, and their exploratory behavior
ceases ahead of schedule. In some cases,
SMCs make a precocious, stable attachment
to the animal pole (Fig. 3; also see Hardin
and McClay, 1990). SMCs make transient
contacts with other areas of the blastocoel
wall when they are indented, but the archen­
teron continues past such indentations,
eventually attaching to the usual site (Har­
din and McClay, 1990). Second, when con­
tact of SMCs with the animal pole is pre­
vented by extruding embryos into capillary
tubing so that filopodia extended by SMCs
cannot reach the animal pole, SMCs con­
tinue the cyclical extension of filopodia for
abnormally long periods of time (Hardin
and McClay, 1990). If the archenteron is
prevented from reaching the animal pole for
several hours, some SMCs detach from the
archenteron, migrate to the animal pole, and
TARGET RECOGNITION BY MESENCHYME DURING SEA URCHIN GASTRULATION
363
Release
Elongate
/
No release
Dent at
uteral dent
Q=-.-- at
FIG. 3. Experiments by Hardin and McClay (1990) demonstrating the presence of an animal pole "target" for
secondary mesenchyme cells (adapted from McClay et al., 1992, with permission). Indentation of the animal
pole with a micropipette results in precocious attachment of the tip of the archenteron to the animal pole region;
elongation of an embryo in capillary tubing prevents attachment of the archenteron to the animal pole. In
elongated embryos, some SMCs migrate as individuals to the animal pole, where they aggregate (arrow). Bars
= 20 /-Lm.
undergo the change in behavior seen in nor­
mal embryos (Fig. 3; Hardin and McClay,
1990). Thus, SMCs appear to be pro­
grammed to continue extending filopodia
until the appropriate target is reached on
the wall of the blastocoel.
The animal pole region of the embryo
focuses filopodial attachments as gastrula­
tion nears its conclusion. Since filopodia can
attach to the animal pole several hours
before they typically do and for several hours
after they normally do, target recognition
allows for temporal flexibility during gas­
trulation. Target recognition by SMCs has
other consequences as well: the animal pole
region and archenteron bend towards the
ventral (oral) side of the embryo after gas­
trulation (Gustafson and Kinnander, 1960;
Dan and Inaba, 1968), thereby moving the
archenteron close to the site where it fuses
with the stomodeum .
Spatial extent of the target
Based on time-lapse cinemicrography, the
region to which SMCs respond is fairly small,
on the order of 20 ~m (Hardin and McClay,
1990), and appears to correspond well with
the region later occupied by the oral attach­
ment that generates the mouth. More pre­
cise mapping can be performed to establish
the spatial extent of the target region by
transplanting rhodamine-labeled SMCs into
unlabeled hosts. When cells are removed
from the tip of the archenteron of a labeled
donor embryo and transplanted into a host
midgastrula, the transplanted cells migrate
and take part in apparently normal pattern
forming events. Discrete clusters of the
364
JEFF
HARDIN
FIG. 4. Spatial extent of the animal pole target revealed by transplants of rhomdamine-Iabeled SMCs into
unlabeled host embryos. (a) bright field, (b) epifiuorescence. RITC-Iabeled cells aggregate in a focal spot near
the animal pole (arrow). Bar = 20 I'm.
transplanted cells form a rosette pattern near
the animal pole, in precisely the location
where the archenteron attaches at the end
of gastrulation (Fig. 4). This region is quite
smaIl, on the order of 15-20 jlm, agreeing
well with the previous estimates based on
differences in filopodial behavior.
Phylogenetic variations in target
placement and utilization during
gastrulation
It seems clear that the shape of the embryo
at the onset of gastrulation can impose sig­
nificant constraints on how gastrulation
progresses. In the normal Lytechinus var­
iegatus embryo, successful filopodial at­
tachments are restricted to lateral regions of
the embryo early in gastrulation (Trinkaus,
1984; Hardin and McClay, 1990). Numer­
ous experiments indicate that this is due to
the shape of the embryo; the animal pole is
simply too far from the tip of the archen­
teron for SMCs to make successful attach­
ments to it (Hardin and McClay, 1990).
However, a second morphogenetic process,
autonomous extension of the archenteron,
operates concurrently with the onset of fil­
opodial exploration (Hardin, 1988, 1989).
As the archenteron extends, the probability
of a filopodium/target encounter is greatly
enhanced by the proximity of the animal
pole to the tip of the archenteron at the 2/3­
% gastrula stage. As stable filopodia remain
attached they puB the archenteron even
closer towards the animal pole, thereby giv­
ing more filopodia opportunities to make
stable contacts with the animal pole region.
By combining a small set of relatively sim­
ple ceIl behaviors (i.e., autonomous cell
rearrangement, random exploration by fil­
opodia, and target recognition), sea urchin
gastrulation is a "robust" process that can
be successfully completed despite wide vari­
ations in embryonic shape and the positions
of interacting tissues.
A survey of gastrulation in a number of
sea urchin species reinforces this notion. Dif­
ferent species rely on varying combinations
of these processes during archenteron elon­
gation, and these differences appear to arise
in part from differences in embryonic shape
(Fig. 5; Hardin and McClay, 1990). Modes
of archenteron elongation include "central
elongation," in which the archenteron is
equidistant from all lateral ectodermal sur­
faces (e.g., Lytechinus variegatus, L. pictus),
"dorsal crawling," in which the dorsal ecto­
derm is near the tip of the archenteron
(Psammechinus miliaris, Echinus microtu­
berculatus), and "ventral crawling," in which
the ventral side is closer (e.g., S. purpura­
IUS). In still other species, such as the cida­
roid, Eucidaris tribuloides, and the Japanese
sand dollar, Clypeaster japonicus, filopodia
may extend laterally towards the stomodeal
invagination directly, in a manner similar
TARGET RECOGNITION BY MESENCHYME DURING SEA URCHIN GASTRULATION
v
D
v
D
Ventral crawlers
( Strongylocentrotus)
Central elongation
( Lytechinus )
v
365
D
Dorsal crawlers
(Echinus, Psammechinus )
V?
D?
Central elongation
( Eucidaris , M espilia )
FIG. 5. Phylogenetic differences in archenteron elongation and oral contact among various species ofeuechinoids
and cidaroids (from Hardin and McClay, 1990, with permission). V = ventral, D = dorsal.
to mouth formation in asteroids. In all of ila (Boulay el al., 1987; Alberga el aI., 1991;
these cases, the simple cell behaviors that Leptin, 1991; Ip el al., 1992; Whiteley el
are responsible for archenteron elongation . al., 1992), Xenopus (Sargent and Bennett,
and attachment appear to be flexible enough 1990), zebrafish (Hammerschmidt and
to permit such phylogenetic variations in Niisslein-Volhard, 1993; Thisse et aI., 1993),
embryonic shape.
chick (Nieto el al., 1994) and mouse (Nieto
el al., 1992; Smith el aI., 1992). Based on
A SEA URCHIN HOMOLOGUE OF THE sequence analysis, the only highly con­
DROSOPHILA PROTEIN SNAIL Is served region of the molecule in all of these
EXPRESSED BY MESENCHYME cases is the putative DNA binding domain,
Recently, we have identified another pop­ which contains five zinc finger loops in all
ulation of patterned mesenchyme by of the species examined. In zebrafish there
searching for sea urchin orthologues of appear to be two snail-related proteins, one
mesodermal transcription factors expressed of which lacks conservation ofthe first loop
in phylogenetically diverse organisms. One (Thisse el al., 1993); in mice, the protein
such transcription factor is snail. Members characterized so far only possess four loops
of this family of zinc finger proteins have (Nieto el al., 1992; Smith el al., 1992). We
been characterized extensively in Drosoph­ have used the DNA binding domain of the
366
JEFF HARDIN
Drosophila snail orthologue to clone and
characterize a sea urchin member of this
gene family (Fig. 6a) Based on whole mount
in situ hybridization, sea urchin snail (usna)
transcripts are first detectable midway
through gastrulation in the archenteron. By
the end of gastrulation, a cluster of cells in
the tip of the archenteron, disposed asym­
metrically with respect to the right-left axis
of the embryo, express usna transcripts.
After gastrulation, two clusters of 6-10 mes­
enchyme cells express usna transcripts near
the sites where the arm buds will grow
shortly after gastrulation (Fig. 6b). By the
prism stage, staining disappears at the tip
ofthe archenteron, and intense staining per­
sists in the two clusters, but eventually dis­
appears by the pluteus stage. It is not clear
whether cells expressing usna are a subset
of PMCs or a subpopulation of SMCs that
have left the vegetal plate or the archenteron
during gastrulation. Interestingly, staining
of what appears to be the same clusters is
obtained for the myogenic factor SUM -1 (J.
Venuti, personal communication). What
function these clusters ofcells play, whether
or not they arise by migration of cells away
from the tip of the archenteron, and how
their bilateral pattern is produced are
unknown at present.
Wilt, 1990; Davidson, 1989). It is also clear
that local cell-cell interactions between cells
from different tissue territories can influ­
ence the expression of particular cell fates
in dramatic ways.
One well-studied example of cell inter­
actions during early sea urchin developmen t
is the influence of micro meres on the devel­
opment of cells with which they are in con­
tact. Two potential effects of micro meres on
nearby cells have been documented. First,
by studying the development of isolated
mesomeres, Wilt and colleagues have shown
that mesomeres can respond to contact with
micromeres by producing gut and skeletal
structures (see the article in this volume by
Wilt et al., 1995). Second, transplants using
16- and 32-cell embryos by Horstadius
(1935) and Ransick and Davidson (1993)
have shown that micromeres in contact with
meso meres can induce presumptive ecto­
derm to form an archenteron, even though
they do not normally do so. In both sorts
of experiments, it has also been shown that
the induced tissues express the appropriate
marker mRNAs and/or proteins (Khaner
and Wilt, 1990; Ransick and Davidson,
1993; see the article by Wilt et al., 1995).
In the case of micromere transplants into
the animal poles of recipient embryos, both
Horstadius (1935) and Ransick and David­
son
(1993) have shown that an additional
CELL INTERACTIONS REGULATE THE consequence
of the implantation of the
ApPEARANCE OF ECTODERMAL ectopic
micromeres
is the production ofone
PATIERN ELEMENTS or more supernumerary skeletal elements.
Macromere descendants can induce new
In the carefully controlled studies of Ran­
patterning sites for PMCs
sick and Davidson (1993), a complete, bilat­
The pattern information associated with eral skeleton can form straddling the ectop­
the ectoderm during gastrulation in the sea ically induced archenteron. The results of
urchin embryo depends on events during Ransick and Davidson suggest several pos­
early development. Studies by Horstadius sible mechanisms by which these appar­
(1935; reviewed in Horstadius, 1939, 1973) ently novel patterning sites arise: (1) they
and Cameron and colleagues indicate that could arise via inductive signals produced
the major tissue territories of the embryo by the micromeres; (2) they could arise via
arise from founder cells that are born by the sequential inductions, in which presump­
5th-6th cleavage (reviewed in Cameron and tive gut tissue is first induced, and then the
Davidson, 1991). Equally clear is the famous induced tissues sends lateral signals to induce
ability of sea urchin embryos to exhibit reg­ new patterning sites, or (3) a combination
ulative development when blastomeres from of these mechanisms could be required.
Two pieces of experimental evidence
the early embryo are isolated and recom­
bined (see the article by Wilt et al. in this indicate that the second of these possibili­
volume; 1995 also see reviews by Horstad­ ties is at least sufficient to account for the
ius, 1939, 1973; Wilt, 1987; Livingston and induction ofpatterning sites within the ecto­
TARGET RECOGNITION BY MESENCHYME DURING SEA URCHIN GASTRULATION
367
A •
s na J 1
u sna
x s na
KN'IRFKCOECQKHYSTSMGLSKHRQFHCPA.' .i'CNQEKKTHSCEECGKLYTTIGALKMHIR 300 DS TKYHC POCGKEYSTFGGLSKHRQLHCDA- - - -QNKKTFNCKYCDKEYMSLGALKMHIR 2 46 EA EKFQCNL CSKSYSTFAGLSKH.KQLHCDS- - - -QTRKSFSCKYCEKEYVSLGALKMHIR 1) 0 sna i I THTLPCKCPICGKAFSRPWLLQGHIRTHTGEKPFQCPOCPRSFADRSNLRAHQQTHVDVK 36 0 us n a THTLPCKCKFCGKAFSRPWLLQGHIRTHTGEKPFSCPHCQRAFADRSNLRAHLQTHSEVK )0 6 xsn a SHT~.PCYCKTCGKAFSRPWL'L QGHIRTHTGEKPFSCTHCNRAFADRSNLRAHLQTHSDVK 230 390 snall KYACQVCHKSFSRMSLLNKJi SSSNCTI TI A ­
33? us n a KYSCKSCGKTFSRMSLLNKHEESGCISSGSD
25 9 xs na KYQCKSCSRTFSRMSLLHKHEETGC - - TVAH
FIG. 6. Characterization of a population of cells expressing transcripts of a sea urchin member of the snail
family of proteins. (a) sequence comparison within the zinc-finger domain ofconceptual translations of Drosophila
snail, Xenopus laevis snail (xsna), and Lylechinus variegalus snail (usna). Identical amino acids are shaded. The
arrow marks the start of the first of the five canonical zinc finger loops. (b) Whole mount in silu hybridization
of a L. variegalus prism stage embryo. Intense staining of two clusters of mesenchyme is evident (arrows). Bar =
20 !lm.
derm. First, Horstadius transplanted Nile
blue labeled veg2 cells (the cells that nor­
mally produce the archenteron) to an ectopic
location. This resulted in the autonomous
production of a second archenteron by the
implanted cells; in addition, perturbations
in skeletal morphology, including ectopic
skeletal rods, were noted by Horstadius
(1935). We have repeated and extended
these results by using the chimera technique
devised by Wray and McClay (1988) to
introduce single rhodamine-labeled veg 2
cells or their descendants into unlabeled host
embryos. When such cells are incorporated
ectopically, they produce an additional
archenteron, but also induce two new bilat­
eral sites of spicule formation. The ecto­
derm underneath the new skeletal elements
as well as the PMCs that form them are
unlabeled (i.e., from the host), and so it
appears that lateral induction of host ecto­
derm by the incorporated cell produces two
FIG. 7. Implantation of RITC-labeled macromere descendants results in induction of additional patterning
sites for primary mesenchyme. For methods, see the text. (a) Bright field, showing ectopic, bilateral skeletal
elements (small arrows), and an ectopic archenteron (large arrow). (b) Epifluorescence demonstrates that only
the archenteron cells derive from implanted tissue; the extra skeletal elements and the ectoderm that underlies
them are not labeled, indicating that they arise from induction and morphopgenetic movements of host tissue.
Bar = 20 !lm.
368
JEFF HARDIN
stomodeal
invaginatiO~
IA\~Q
~'"
ball
isolate at
mesenchyme
blastula stage
/l8
lDJectRITC
~.~~
j)
isolate at early
gastrula stage
oral hood
and mouth
~
"reconstituted"
oral hood
oral hood
and mouth
!lQ
FIG. 8. . Summary of experiments demonstrating that the site where oral structures form is fixed by the early
gastrula stage. When animal caps are isolated at the mesenchyme blastula stage, they do not produce a stomodeal
invagination, and a mouth forms via regulation within the vegetal fragment. In contrast, when the isolation is
performed at the early gastrula stage, the animal fragment produces a stomodeal invagination, and can support
normal patterning of PMCs when they are transplanted into it. Conversely, the vegetal fragment is no longer
capable of forming a mouth.
new ectodermal patterning centers (Fig. 7;
Hardin, Benink, and Wray, manuscript in
preparation).
The oral field is restricted to its normal
site by the early gastrula stage
The final skeletal pattern produced by the
sea urchin larva is considerably more com­
plex than two bilaterally disposed rods. The
pattern produced at various locations within
the larva is regionally specific as well as spe­
cies-specific (Ettensohn, 1990; Armstrong
and McClay, 1994). How are regional aspects
ofskeletal pattern elaborated? Although final
conclusions cannot yet be drawn regarding
the entire pattern that is produced, several
experiments indicate how and when pattern
information is laid down in the oral region
of the larva. Again, classical experiments by
Horstadius hint at cell interactions that are
important. He found that the cells destined
to give rise to most of the ectoderm (the ani
and an 2 tiers in his terminology) formed
Dauerblastulae when isolated at the 32- or
64-cell stage. However, when the next more
vegetal (veg l ) tier is included, the an/an 2
progeny form a stomodeal invagination (the
ectoderm's contribution to the mouth; Hor­
stadius, 1935). This suggests that interac­
tions between the veg l progeny and adjacent
tiers result in induction of the oral field.
More recently, we have performed addi­
tional isolations to address this question
(Hardin and Armstrong, manuscript in
preparation). By isolating animal poles at
progressively later stages, we have been able
to determine the time during which the pat­
tern information resident in the oral region
becomes fixed. When the animal pole is
removed prior to the early gastrula stage,
the remaining vegetal tissue can regulate to
produce a new site of mouth formation,
whereas the animal hemisphere does not
produce a stomodeum. However, if the
experiment is performed somewhat later, at
the early gastrula stage, the animal pole frag­
ment produces a stomodeal invagination,
and the remainder of the embryo is unable
to produce a mouth. Transplantation of
TARGET RECOGNITION BY MESENCHYME DURING SEA URCHIN GASTRULATION
PMCs into animal hemispheres isolated at
various times indicates that the pattern
information required for the production of
the parallel skeletal rods flanking the mouth
is coordinately regulated with the oral field
(these experiments are summarized in Fig.
8). When isolated at progressively later
stages, there is a sharp increase in the ability
ofanimal pole fragments to support normal
patterning (Hardin and Armstrong, in prep­
aration).
CONCLUSIONS
The studies we have performed allow us
to draw several conclusions regarding how
mesenchymal cells find their targets in early
embryos. First, in many cases interactions
between mesenchyme cells and their targets
appear to be highly local. In the case of
SMCs, where filopodial dynamics have been
examined carefully, these local interactions
appear to be contact-mediated, although our
experiments do not rule out short-range dif­
fusible or graded signals (see Hardin and
McClay, 1990 for further discussion). Sec­
ond, target recognition is a specific cell inter­
action, rather than a general adhesive event.
This conclusion is based on the lack of
attachment of several other mesenchymal
cell types to the animal pole targets for PMCs
and SMCs we have identified, and the very
different ways in which these two popula­
tions of cells interact with the ectoderm of
the oral field. Third, successive cell inter­
actions between founder cells and their
progeny in the early embryo appear to be
responsible for establishing the target regions
we have identified, at least in the case of
two large clusters of PMCs in the ventro­
lateral regions of the embryo and mesen­
chyme in the oral region. The inductive and!
or inhibitory events associated with the
establishment of such pattern information
has not been characterized, although estab­
lished signal transduction pathways within
the cell will undoubtedly be involved (see
the article by Wilt, 1995; Cameron et al.,
1994). The specific molecular basis for the
guidance cues we have identified remains
unclear. Putative receptor-ligand interac­
tions that might mediate specific target rec­
ognition events await identification at the
369
molecular level. Nevertheless, the cellular
interactions we have identified serve as an
experimental framework defining processes
that candidate guidance and regulatory mol­
ecules may control.
ACKNOWLEDGMENTS
This work was supported by a Lucille P.
Markey Scholar Award in the Biomedical
Sciences, a NSF grant and by a NSF Young
Investigator Award to the author.
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