3.

The Sea Urchin

leffHardin

Introduction
The sea urchin embryo has been used for more than a century mental biology, and the importance and relevance of such
to study many problems central to developmental biology. experiments today is referred to Wilt (1987), Davidson (1989;
During the latter part of the nineteenth century, marine stations 1990) and Livingston and Wilt (1990).
in Italy, France, and the United States flourished, and the More recently, the sea urchin embryo has been used as a
embryos of marine organisms were found to be favourable convenient system for studying morphogenetic movements and
material for investigating early embryonic development. The cell interactions during gastrulation, the changes in gene
study of echinoderms and, in particular, of sea urchins, that expression associated with the establishment of tissue territories
was carried out at these marine stations was influential in the along the embryonic axes, and phylogenetic variability and
formation of many seminal ideas in developmental biology associated modifications in early development. In summarizing
(for reviews, see the classic texts of Wilson, 1925; Morgan, such work, this chapter provides a brief overview of normal
1927). Later, in the early part of the twentieth century, the development in the sea urchin embryo and a few case studies
experiments performed on sea urchin embryos using chemical illustrating modem uses of this system for studying early develop­
agents and the classic blastomere recombination experiments mental events. Where possible, the reader is referred to reviews
performed by Horstadius (1939; 1973) paved the way for ideas that treat individual topics in more detail than is possible here.
about graded distributions of morphogenetic substances in the For methods of maintaining adults, obtaining gametes, and
embryo. The sea urchin also provided useful material for culturing embryos, see Hinegardner (1967; 1975a) and Leahy
studying many aspects of nucleic acid structure, complexity, (1986). General methods for culturing and experimentally
and function in the early days of molecular biology (reviewed manipulating embryos can be found in Harvey (1956) and
by Davidson, 1988). The reader interested in the historical role Horstadius (1973), while more up-to-date methods are in
played by sea urchin embryos in the emergence of develop- Schroeder (1986).

Normal development
Sperm, eggs, and fertilization
Sea urchin and sand dollar gametes can be obtained in large by Schroeder (1980b; see also Maruyama et al., 1985).
numbers by intracoelomic injection of 0.5M KCl or by Another marker of polarity in the unfertilized egg is the sub­
electrical stimulation; this leads to the shedding of gametes equatorial concentration of orange pigment in some batches of
into sea water (in the case of eggs) or 'dry' into a dish (in the eggs of the Mediterranean sea urchin, Paracentrotus lividus,
case of sperm). Depending on the species, several millilitres of especially those obtained from Villefranche in France (Boveri,
ripe eggs or sperm can be obtained from a single animal and 1901; Horstadius, 1973; Schroeder, 1980a).
the embryos can be conveniently reared in finger bowls or in Unfertilized eggs possess several other kinds of distinct
stirring cultures. The major stages of early development in the granules with different distributions within the egg. Cortical
sea urchin are shown in 3.1-3.15, and each stage will be granules lie immediately beneath the egg surface and are
discussed in turn in the following sections. Mature sea urchin released upon fertilization. These are lamellar structures which
eggs, unlike eggs from many other animals, have completed contain components necessary for the construction of the
meiosis and the extrusion of polar bodies in the ovary to fertilization envelope (3.2) and the hyaline layer, an
produce a haploid gamete (3.1). Immediately apposed to the extracellular matrix layer which lies on the outside of the
egg plasma membrane is the vitelline envelope which contains embryo. Pigment granules are particularly prominent in
the glycoproteins essential for species-specific fusion of sperm species such as Arbacia punctulata from America. Other
and egg, while freshly shed eggs are surrounded by a jelly granules, which release their contents following fertilization,
coat. Marking the jelly coat with small ink particles reveals the but on a much slower time course than the cortical granules,
jelly canal, a marker for the animal pole of the egg first contain extracellular matrix proteins. Some of these granules
described by Boveri (1901) and more recently re-investigated can be redistributed by centrifuging eggs suspended in sucrose

37
3.1 3.2 3.3

3.4 3.5 3.6

3.7 3.8 3.9

3.10 3.11 3.12

38
 3.13 3.14 3.15

3.1-3.15 Characteristic stages during early development in LytechinU5 variegatu5 . The animal pole, when evident, is at the
top unless otherwise indicated . 3.1, unfertilized egg; 3.2, recently fertilized egg (note the fertilization envelope and the
fertilization cone, at the top of the egg); 3.3; two-cell; 3.4, 4-cell; 3.5, 8-cell; 3.6, 16-cell (the micromeres are at the bottom) ;
3.7, 16-cell embryo viewed from the vegetal pole to show the micromeres; 3.8, 32-cell embryo (the small micromeres are
visible at the extreme vegetal pole); 3.9, vegetal pole view of a 32-cell embryo showing the small micromeres and the larger
micromere derivatives immediately above them; 3.10, blastula stage; 3.11, mesenchyme blastula; 3.12, early gastrula
(courtesy of C. Ettensohn); 3.13, late gastrula; 3.14, prism stage embryo (note the coelomic pouches beginning to form) ; 3.15,
pluteus (x450) .

density gradients with little effect on patterns of development actin filaments in the egg cortex produces the fertilization cone
(reviewed in Harvey, 1956; McClay et al., 1990; 3.16). (3.17). The fast block to polyspermy, a rapid depolarization of
Under normal circumstances, sea urchins simply shed the egg mediated by an influx of sodium ions, provides an initial
gametes directly into the marine envirorunent. Three mechanisms barrier to penetration of the egg by more than a single sperm. A
appear to help ensure that interactions between sperm and egg are slower but more permanent block to polyspermy results from
species-specific. First, in Arbacia punctulata, the peptide resact, cortical granule exocytosis and elevation of the f ertilization
which is contained in the egg jelly, appears to be a species­ envelope (3.2) which is triggered by release of calcium ions in a
specific chemo-attractant for sperm (Ward et al., 1985). Second, wave that sweeps across the egg. Following its entry into the
activation of sperm by egg jelly is also species-specific in some egg, the sperm nucleus decondenses to form the male
species so that contact between them results in a rapid acrOsome pronucleus. Microtubules polymerize away from the sperm
reaction . The acrosomal process which contains proteolytic centriole in the direction of the female pronucleus, the two
enzymes, fuses with the sperm plasma membrane and extends pronuclei migrate towards one another, and ultimately they fuse
dramatically, driven by the rapid polymerization of actin to form the diploid, zygote nucleus (Bestor and Schatten, 1981).
microfilaments. Third, the adhesion of activated sperm to the Ultimately, the release of calcium responsible for cortical
vitelline layer is mediated by the acrosomal protein bindin, whose granule exocytosis results in activation of the egg, a complex
binding to the vitelline envelope also appears to be mediated by a series of events leading to the initiation of protein and DNA
species-specific ligand-receptor interaction (Glabe and Vacquier, synthesis. Egg activation can be mimicked by a number of
1978; Moy and Vacquier, 1979). All regions of the egg surface treatments which lead to parthenogenetic activation and include
support sperm attachment and fusion in the sea urchin. treatment with butyric acid and hypertonic sea water and
At the site of sperm-egg fusion , localized polymerization of calcium ionophores (reviewed by Weidman and Kay, 1986).

Cleavage and the blastula

Sea urchins undergo synchronous, radial, holoblastic cleavages separating the embryo into an animal and vegetal quartet of
until the blastula stage. The first two cleavages are meridional cells (3.5). At the fourth cleavage, the cells of the animal tier
and perpendicular to one another, passing through the animal divide equally to produce eight mesomeres, while the cells in
and vegetal poles to produce first two and then four cells of the vegetal tier divide unequally , producing four large
equal size (3.3-3.5 and 3.18). The third cleavage is equatorial, macromeres, and four small micromeres (3.6, 3.7 and 3.19). In

10
 3.16 3.17

3.18 3.19

3.20 3.16-3.20 An Arbacia punctulata egg stratified by

centrifugation in a sucrose gradient is seen in 3.16
(x500). Red pigment granules stratify at one end of
the egg. 3.17 (x20,000) shows a scanning electron
micrograph of a sperm entering a Lytechinus pictus
egg. The vitelline layer, with microvilli protruding
into it, is also visible (courtesy of G. Schatten). 3.18
(x1500) shows first mitosis visualized by double
labelling of microtubules in the spindle (orange) and
DNA (blue) in Lytechinus pictus (courtesy of J.
Holy). 3.19 (x600) shows asymmetric division of an
isolated vegetal pole blastomere into a macromere
and a micromere at fourth cleavage. 3.20 (x700)
shows a macromere/micromere pair double labelled
for microtubules (green) and DNA (blue) to show the
asymmetric placement of nuclei and spindles during
fourth cleavage (courtesy of J. Holy).

the vegetal blastomeres of the 8-cell embryo, the nucleus and the macromeres divide meridionally to produce a tier of eight
moves to an eccentric, vegetal position, and the mitotic spindle 'half-macromeres'. The micromeres divide asymmetrically to
is subsequently assembled eccentrically as well, with the result produce a tier of small micromeres at the extreme vegetal pole of
that the aster is flattened and shortened (3.20). The asymmetric the embryo, and a tier of larger micromere derivatives im­
fourth cleavage is the first sign that cells distributed along the mediately above them (3.8, 3.9). At the sixth cleavage, all cells
animal-vegetal axis of the embryo are different and only the divide equatorially to produce a 64-cell embryo with five tiers: the
micromeres will go on to form primary mesenchyme cells daughters of the an] and an 2 cells lie at the animal pole, the veg l
(pmc) that produce the larval skeleton (see below). and veg 2 tiers, derived from the macromeres, lie in the vegetal
At the fifth cleavage, the mesomeres divide equatorially to hemisphere, and the micromere descendants lie at the vegetal
produce two animal tiers (denoted by Hbrstadius as anI and an 2), pole. At the seventh cleavage, all cells divide meridionally to

40
produce a 128-cell blastula. During the blastula stage, cells no embryonic environment from the external environment, with
longer cleave synchronously: as development proceeds, divisions the epithelium becoming impermeable to small sugar
of local groups of cells remain synchronous, but these regions molecules by the midblastula stage (Moore, 1940). The forces
gradually decrease in size, and eventually the cell cycle lengthens responsible for formation of the blastocoel remain unknown,
and becomes largely randomized (Dan et at., 1980). although osmotic influx of water into the blastocoel and
The early blastula is an epithelial monolayer enclosing a attachment to the hyaline layer have been suggested as
central , spherical blastocoel (3.10) whose cells develop septate possible factors (Dan, 1960; Dan and Inaba, 1968; Gustafson
junctional contacts between one another (Spiegel and Howard , and Wolpert , 1962). At the midblastula s tage, each cell
1983) and begin to produce the basal lamina lining the produces a cilium, the embryo begins to rotate within the
blastocoel. The exterior, apical ends of the epithelial cells fertilization envelope, and a hatching enzyme is synthesized
possess numerous microvilli , which are embedded in the that is secreted into the space between the embryo and the
hyaline layer, and apical lamina (Hall and Vacquier, 1982). fertilization envelope. Here it digests the envelope and allows
The cells of the blastula eventually seal off the internal, the embryo to hatch and become a free-swimming blastula.

Establishment of the major tissue territories of the embryo

What changes in the shape of the embryo take place during stage, gives rise to the ectoderm of the animal pole, the an 2 tier
gastrulation, and what consequences do they have for sub­ gives rise to equatorial ectoderm , the veg j tier of the 64-cell
sequent tissue-specific differentiation? Analyses of cell fate embryo gives rise to the vegetal pole ectoderm and the veg 2
and cell movements both shed light on these questions . By tier generates the cells of the archenteron and a group of
staining individual blastomeres with Nile blue at the 32- and mesenchymal cells, the secondalY mesenchyme, which form at
64-cell stages, Horstadius (1935) constructed a fate map for the tip of the archenteron, while the large micromere
the Paracentrotus li vidus embryo (3.21). According to derivatives give rise to the primary mesenchyme which forms
Horstadius, the ani tier of cells, which is distinct at the 32-cell the skeletal structures of the larva.

3.21 3.22
3.21-3.24 Organization and

fates of cells in the early sea onl ~

urchin embryo according to animal .( •• ~ ••• ~ •••r'p.~
Horstadius (for Paracentrotus cop : ••• : •••: : •• :
lividus; 3.21) and Cameron
and co-workers (Strongylo­ . . ,,·1.. .. \....

on 2 . . .
•••••••• • 1_ ..... ••: •

•.• t·
: • e. • .. .
XXX X I.­
centrotus purpuratus; coloured li / ~ X X
diagrams ; from Davidson , vegl ~'x Xx <x : :
u X xx X ~(x x"x-{
1988, with permission). The // / \ \
coloured regions refer to the veg 2 I( \ I J }
\l"",.>_._L/
following presumptive tissue skel. B sm. mic /
territories. (Key : red = skele­
togenic cells, yellow = ab­
oral (dorsal ) ectoderm, green
= oral (ventral) ectoderm,
magenta = small micromere 3.23 3.24
derivatives, blue = archent­
eron and associated structures.)
apical
tuft

mouth
midgut intestine
anus foregut

midgut

larval i=====
skeletogenic skeleton
mesenchyme
cells

41
3.25 3.26 3.27

3.28 3.29 3.30

3.31 3.32 3.33

3.25-3.33 Examples of tissue-specific gene expression in the sea urchin embryo. Tbe probes described here are representative,
but by no means exhaustive. 3.25 (x450) is a whole mount in situ hybridization using an anti-sense BP-10 RNA (courtesy of T.
Lepage; Lepage et at., 1992). The boundary of expression sharply demarcates future ectoderm from endoderm and mesoderm.
3.26 (x420) shows an in situ hybridization of a Lytechinus variegatus embryo using an anti-sense probe for LvS1 (Wessell et at.,
1987), a member of the Spec 1 family of genes (Lynn et at., 1983 ). 3.27 (x350) shows a section of a Lytechinus variegatus
pluteus immunostained for the Ecto V antigen (V for ventral) ; staining is complementary to aboral markers (courtesy of D.
McKlay; Coffman and McClay, 1990).3.28 (x450) shows whole mount immunostaining for the msp130 homologue in
Lytechinus variegatus, a cell surface antigen expressed by primary mesenchyme cells and one of a host of probes specific to
these cells (e.g., Benson et at., 1987; Drager et at., 1989 ; Leaf et at., 1987; Wessell and McClay, 1985). 3.29 (x350) shows
pigment cells localized to the presumptive arm buds of a Lytechinus variegatus prism stage embryo. 3.30 (x350) shows
immunostaining for Meso 1, a cell surface epitope present on the surfaces of primary and secondary mesenchyme cells (Wray
and McClay, 1988). 3.31 (x400) shows immunostaining for Endo 1, an antigen expressed by the mid- and hindgut of the
archenteron (Wessell et at., 1985).3.32 (x200) shows in situ hybridization using an anti-sense probe for LvN1.2, which also
localizes to this region (courtesy of G. Wessell; Wessell et at., 1989).3.33 (x500) shows rhodamine-phalloidin staining for the
esophageal muscle bands which surround the foregut (Ishimoda-Takagi et at., 1984; Wessell et at., 1990).

42
 More recently, these lineage studies have been refined and when there are approximately 64 cells in the embryo
extended to account for distinctions along the dorsoventral (3.22-3.24). The four animal blastomeres of the 8-cell embryo
axis of the early embryo. Based on unique patterns of gene contribute progeny to either oral or aboral ectoderm, and they
expression, cell lineages, and one or more characteristic are termed Na and No cells (animal, oral and aboral). Some
differentiated cell types, sea urchin embryonic cells can be cells from Horstadius' veg 1 tier, derived from the macromeres
classified into five major tissue territories (3.22-3.24; of the 16-cell embryo, form aboral ectoderm, while others
Cameron and Davidson, 1991; Davidson, 1989). These are: contribute to oral ectoderm. The veg 2 tier of Horstadius gives
rise to the structures of the archenteron : the larger (animal)
The aboral (or dorsal) ectoderm , which forms a simple,

progeny of the micromeres generate skeletogenic mesen­

squamous epithelium.

chyme, whereas the small (vegetal) progeny of the micromeres

The oral (or ventral) ectoderm, which forms the epithelium

contribute to the coelomic pouches.

of the mouth region and the ciliated band, a structure that

The clonal boundaries described by Cameron and co-workers

lies at the boundary between oral and aboral ectoderm.

are established through invariant cleavages and these boundaries

The vegetal plate, which gives rise to the archenteron and

seem to coincide very closely with spatially restricted patterns of

its derivatives.

gene expression (3.25-3.33). That clonal boundaries and patterns

The primary mesenchyme cells, which will produce the

of gene expression are co-extensive suggests that the reliability

larval skeleton.

of cleavage may be important in establishing patterns of

The small micromeres, which have been reported to

differentiating tissue within the unperturbed embryo. The sea

contribute to the coelomic pouches (Pehrson and Cohen ,

urchin embryo is, however, well known for the ability of its cells
1986).

to adopt new fates when placed in unusual environments,

Each of these tissue territories derives from a specific group of indicating that the establishment of reliable clonal boundaries
founder cells whose lineages become distinct during cleavage. does not reflect an underlying 'mosaic' quality of the early cells
The lineages of cells lying within these domains can be of the sea urchin embryo, in contrast to some other invertebrate
distinguished by the completion of the sixth cleavage, i.e. embryos (e.g. , nematodes and ascidians).

Gastrulation and post-gastrula development

The various territories of the embryo also differ in their the blastocoel for their migration (Lane and Solursh , 1988;
patterns of motility as the embryo is transformed during Lane and Solursh, 1991 ; Solursh et al., 1986). Primary
gastrulation. The study of sea urchin gastrulation has been mesenchyme cells migrate away from the vegetal plate, but
influential in shaping ideas about general mechanisms of eventually form a ring in the vegetal pole region of the embryo
morphogenetic movements, and the work of Gustafson and co­ (3.35) . Ultimately, two clusters of pmc form in the ventro­
workers in particular demonstrated the power of time-lapse lateral ectoderm and give rise to the spicule rudiments of the
microscopy in elucidating morphogenetic processes (reviewed larva (3.36; for further details, see Solursh, 1986, and Decker
by Gustafson and Wolpert, 1963; 1967). Just prior to and Lennarz, 1988).
gastrulation , there is a dramatic decrease in the overall rate of Following the ingression of pmc, pigment cells depart from
cell division, and the embryo comprises about 1000 cells. At the vegetal plate (Gibson and Burke, 1985; 3.30). The vegetal
the animal pole, a thickened region of epithelium, the apical plate then begins to bend inward to form a short, squat
plate, or acron, appears with a tuft of cilia that are longer than cylinder, the archenteron. During this initial phase of
those found on the rest of the embryo (3.11), while the invagination (primary invagination), the archenteron extends
I
epithelium at the vegetal pole of the embryo flattens and 1/C / 2 of the way across the blastocoel (3.12). A short pause
thickens to form the vegetal plate. follows primary invagination , after which the archenteron
The onset of gastrulation is marked by the ingression of resumes its elongation (secondary invagination). At about the
primary mesenchyme cells (pmc) into the blastocoel (3.11 and time secondary invagination begins, cells at the tip of the
3.34-3.36), an event accompanied by alterations in cell archenteron (secondary mesenchyme cells) become protrusive,
polarity and loss of the epithelial phenotype (Anstrom and extending long filopodia into the blastocoel (3.13). Eventually
Raff, 1988) and the appearance of new cell-surface deter­ the archenteron elongates across the blastocoel , and its tip
minants and transcripts (see above). When they begin to attaches to the ventral ectoderm near the animal pole.
ingress, pmc become bottle-shaped in profile as the surface By the time the archenteron completes its elongation, pmc
area of their apical ends is reduced (Katow and Solursh, 1980), have localized into two major clusters in the ventrolateral
and they eventually detach from the hyaline layer. These cells ectoderm to form spicule rudiments . At the tip of the
use bristle-like filopodia to move (reviewed by Solursh, 1986) archenteron, two bilateral outpocketings, the coelomic
and require sulphated proteoglycans on their surfaces and/or in pouches, appear (3.14). Ultimately the tip of the archenteron

43
3.34 3.35

3.36 3.34-3.36 Primary mesenchyme cell behaviour during early

development. 3.34 (xSOO) is a scanning electron micrograph
of a Lytechinus variegatus mesenchyme blastula during
ingression of primary mesenchyme cells (courtesy of ].
Morrill; magnification . 3.35 (xSOO) is an L. variegatus gastrula
viewed from the vegetal pole , showing the aggregation of
primary mesenchyme cells into two ventrolateral clusters
(magnification. 3.36 (x2S0) is an L. variegatus pluteus viewed
with darkfield optics (courtesy of M.A. Alliegro; magnifica­
tion.

fuses with the ectoderm to form the larval mouth . As the echinus rudiment, the imaginal structure which generates the
pluteus larva develops (3.15), the archenteron becomes juvenile urchin during metamorphosis (Czihak, 1971;
tripartite, and a host of differentiated tissues appear that Okazaki, 1975a). The eversion of the echinus rudiment is a
include nerve cells in the ectoderm (Bisgrove and Burke, geometrically complex process and the interested reader is
1987). The left coelomic pouch ultimately forms the hydrocoel urged to consult the beautiful drawings found in Czihak (1971)
which, together with the ectodermal vestibule, gives rise to the and Okazaki (1975a).

The sea urchin as an experimental system

Mutational analysis is not practical in the sea urchin because of genetics is feasible, however, the early embryonic development
the long generation times and the difficulties of rearing embryos of the sea urchin can be manipulated directly , thereby allowing
through metamorphosis in the laboratory (but see some epigenetic influences on development to be examined. In
interesting mutants produced by Hinegardner, 1975b). In contrast particular, the sea urchin system is an excellent system in which
to many of the commonly used experimental systems in which to study the role of cell interactions during early development.

Cell interactions in the early embryo

The landmark experiments of Driesch (e.g., 1891) in which he normal larval tissues. Since that time, the sea urchin embryo has
separated early blastomeres of the sea urchin embryo been used to study how cells become committed to various
demonstrated that cells derived from the 2- and 4-ceJl embryo pathways of differentiation . In particular, some of the most
could regulate to produce a small embryo possessing most of the remarkable experiments in the history of embryology were those

44
performed by Horstadius (1939, 1973) investigating the they produce spicules and archenterons (Henry et al., 1989).
properties of cells along the animal-vegetal axis of the early sea Pervasive alterations in zygotic gene expression also result
urchin embryo. By separating unfertilized eggs and early when cell contacts are continuously prevented from forming
embryos into animal and vegetal halves, separating tiers of cells by stirring blastomeres in calcium-free sea water (Hurley et
at the 32- and 64-cell stages, and by juxtaposing various tiers of al., 1989; Stephens et al., 1989). Cell contact and cell
cells originating at different locations along the animal-vegetal interactions thus seem to be important in specifying cell fate in
axis, Horstadius found that a graded influence on differentiation experimentally treated blastomeres.
existed along the animal-vegetal axis. These experiments led to In contrast to most cells in the early sea urchin embryo,
the idea that a ' vegetalizing gradient', originating in the unferti­ however, micromeres appear to be committed to a spiculo­
lized egg, exerts effects along the embryonic axis. More recently, genic pathway as soon as they appear at the 16-ceU stage. This
micromeres have been shown to interact with meso meres at the conclusion is largely based on the work of Okazaki (1975b),
level of single pairs of cells, so that eventually the mesomeres who developed culture conditions allowing micromeres to
form an array of mesodermal and endodermal structures differentiate in vitro. Cultured micromeres divide, become
(reviewed by Livingston and Wilt, 1990). The effects of this motile, migrate, and the cells later associate into syncitia and
vegetalizing influence can be mimicked by the vegetalising produce spicules (Okazaki, 1975b; see 3.41). They also under­
agent, lithium chloride, which confers progressively more go several simultaneous adhesion changes at the time that pmc
vegetal qualities to blastomeres which would not normally would ordinarily ingress into the blastocoel; these include:
possess them (see Horstadius, 1973). More recent experiments
A loss of an affinity for the hyaline lamina proteins hyalin

using molecular assays have confirmed that lithium induces

and echinonectin.

alterations in gene expression that include overproduction of

A loss of an affinity for neighbouring cells.

endoderm in whole embryos (Nocente-McGrath et al., 1991) and

An increased affinity for the basal lamina (Fink and

the appearance of mesodermal and endodermal markers in the

McClay, 1985; Burdsal et al., 1991).

descendants of single mesomeres (Livingston and Wilt, 1989).

Cell contact also seems to be important in regulating gene That these changes can occur in cultured micromeres suggests
expression and differentiation in the early embryo. When that this transformation is entirely autonomous. The fourth
mesomeres are isolated as an intact group of eight cells from a cleavage can be equalized by treatment of eggs with low
16-cell embryo, they produce a ciliated ball, or Dauerblastula concentrations of sodium dodecyl sulphate and, here, sixteen
(Horstadius, 1939); when , however, mesomeres are dis­ cells of equal size are produced, and, in some but not all cases,
sociated and recombined, thereby altering cell-cell contacts, formation of pmc is suppressed (Langel an and Whiteley, 1985).

Cell interactions and the extracellular matrix

In the forming sea urchin embryo, cells interact not only with 1991), again pointing to an important integrating role for these
each other, but with the extracellular environment. The epithelial apical layers during sea urchin embryogenesis.
tissues of the sea urchin embryo are in contact with several Treatments affecting the basal lamina also block gas­
extracellular matrix layers: the apical lamina, and hyaline layer, trulation. Incubation of embryos from fertilization onward in
on their apical surfaces, and the basal lamina on their basal ~-aminoproprionitrile (BAPN), an inhibitor of lysyl oxidase
surfaces (3.37,3.38). In addition, Dan (1960) has suggested that (an enzyme involved in collagen crosslinking), results in
the hyaline layer is important as a structural support and embryos which develop normally to the mesenchyme blastula
mechanical integrator of epithelia. Consistent with this idea, stage, but fail to progress further. If the drug is removed, even
treatment of sea urchin embryos with monoclonal antibodies after the embryos have been arrested at the mesenchyme
specific for the protein hyalin (a major component of the hyaline blastula stage for more than 24 hours, the embryos begin to
layer) results in visible delamination of the hyaline layer from the gastrulate and complete development normally (Butler et aI. ,
epithelium, abnormal thickening of the epithelium, and blockage 1987; WesselJ and McClay, 1987). In the normal embryo, a
of invagination (Adelson and Humphreys, 1988; 3.39) . If fibrillar meshwork is present in the blastocoel along which
antibody-treated embryos are removed from the antibody, mesenchyme cells appear to migrate (3.40). BAPN treatment
development resumes, and a normal pluteus larva results results in a poorly constructed basal lamina and poor motility
(Adelson and Humphreys, 1988). These results suggest that the of mesenchyme cells (Butler et al., 1987; Hardin, 1987) and,
antibody interferes with the mechanical and structural integrity of in addition, antigens normally expressed in the archenteron fail
the epithelium, but also with more general requirements for the to appear as long as the embryos are incubated with the drug
initiation of gastrulation. Incubation of Strongylocentrotus (Wessell and McClay, 1987). The similarity of the effects
purpuratus embryos from hatching through to gastrulation in Fab observed when either extracellular matrix layer is disrupted
fragments of antibodies which recognize a fibrous apical lamina suggests that a critical period precedes gastrulation during
protein results in disruption of the normal epibolic movements which normal contact with both the basal lamina and the
preceding gastrulation, and failure of invagination (Burke et al., hyaline layer is required in order for gastrulation to begin.

45
 3.37 3.38

3.39 3.40

3.37-3.40 The role of the extracellular matrix during sea urchin gastrulation . 3.37 and 3.38 show
immunofluorescent localization of the hyaline layer component hyalin and a basal lamina antigen, Ibl0
(x550). 3.39 shows the effects of treating a L. variegatus embryo with antibodies against hyalin (x400).
3.40 is a scanning electron micrograph of a protrusion from a secondary mesenchyme cell; thin filopodia
(arrows) contact fibrils of extracellular matrix (xlO,OOO; courtesy of J. Morrill).

Cell interactions between primary mesenchyme cells and surrounding tissues

Although micromeres are committed early in development to contained within the ectoderm which helps specify where pmc
form a pmc (3.41), it is also clear that tbe behaviour of later localize, but the molecular basis of this localized attachment is
cells is affected by the embryonic environment. Okazaki et at. still not understood.
(1962) observed that pmc localize at sites in the ectoderm The pmc not only receive signals from the environment, but
where the epithelial cells are thickened , producing an optical also appear to interact with themselves and other mesenchyme
effect reminiscent of an oriental fan . When this belt of cells is cells . When supernumerary pmc are transplanted into host
shifted along the animal-vegetal axis in vegetalized embryos embryos, the resulting skeleton is indistinguishable from the
treated with lithium chloride, the pmc localize to the shifted normal one, even though as many as two to three times the
ectoderm (Gustafson and Wolpert, 1961; Okazaki et aI., 1962). normal number of cells participate in skeleton formation
Likewise, when the normal differentiation of ectodermal (Ettensohn, 1990b) and it seems that some signal(s) operate
tissues is disrupted by treatment of embryos with NiCl z, pmc which restrict both the size and location of skeletal elements.
form spicules, but in a completely radialized pattern (Hardin et When all pmc are removed from the embryo, a skeleton arises
al., 1992; 3.42), even though transplantation experiments from a subpopulation of secondary mesenchyme cells which
suggest that it is only the ectoderm, and not the mesenchyme become spiculogenic and produce a normal skeleton (Ettensohn
cells, which are affected (Hardin and Armstrong, 1991). These and McClay, 1988; 3.43), even though secondary mesenchyme
experiments imply that regionally specific information is cells do not normally participate in spicule production. The

46
3.41
. \.
. . '). \~. \
·\\1 ..•.\ ' . '0.
3.42

~·fr
t. , .' . .."" " . ...... 4.'
' :.-. .' .

.•~\\'.i-'~
" "'\ . / ""-='';'''''' .

,'., ..... . ./:' :..Y·.

"0/.: .
";" ~ t-"~~'
"'.~~ ,

.?£
';'·l.,\,__
.... ,r::::'
" ';
x',
~ "

~
.... ! oj,' ". '.
1&:' ., • •

·~·f"';.
.... \.~'. _." /'X.. .'\..
:-.... ) • •, / .. 0 '.,_••

'"

' ....... .~.

. '" ' .. ' ... , '.
• '~~ '.1:".... ~~ \~ '\'\.;'.
' . . • it' t" , 0 \ .
~ ~. , . "~.~ 1--:.,..• _. "
0

It

3.41-3.43 Cell autonomy and cell interaction duri ng skele­ 3.43

ton formation in the sea urchin embryo. 3.41 shows five
picules formed by cultured Arbacia punctulata micromeres
(xl SO; courtesy of R. Fink). 3.42 shows radialized skeleton
formed in an embryo treated with 0 .5 mM NiCI 2 from
fertilization through the early gastrula stage (xSOO). 3.43
shows a skeleton produced by 'converted' secondary mes­
enchyme cells in embryos in which all primary mesenchyme
cells were removed (x 100; courtesy of C. Ettensohn) .

pmc must therefore provide some restrictive signal preventing restrictive influence must operate over a distance of tens of
the secondary mesenchyme cells from differentiating into microns since the two populations of mesenchyme are some
spicule-producing cells. This interaction displays a remarkably distance from one another in the embryo. Based on studies by
quantitative character for, as one removes more and more pmc Ettensohn (1990a), the period during which this interaction
from Lytechinus variegatus, a progressively greater number of occurs ends at about the time the archenteron makes contact
secondary mesenchyme cells convert to compensate for the loss with the animal pole (for possible mechanisms, see Ettensohn,
of spiculogenic cells (Ettensohn and McClay , 1988). This 1991).

Cell interactions during archenteron formation

Surprisingly little is known about the forces which promote chondroitin sulphate proteoglycan has been localized to the
the inward bending of the archenteron , although many ideas lumen of the archenteron (Lane and Solursh, 1991). Finally, a
have been put forward. Neither local proliferation of cells nor noticeable amount of epiboly, or spreading of the pre-gastrula
changes in lateral contact between cells which would epithelium, occurs just prior to gastrulation, and this includes
ultimately result in bending seem to be involved (reviewed by tissue immediately adjacent to the vegetal plate (Ettensohn ,
Hardin, 1990). Apical constriction of cells in the vegetal plate 1984; Burke et al" 1991). When these movements do not
has been proposed to account for primary invagination (Odell occur properly, invagination fails, so suggesting that they may
et ai., 1981; reviewed in Ettensohn, 1985b) and there are be important for primary invagination (Burke et at., 1991).
apically constricted cells in the centre of the vegetal plate, with Whatever mechanism(s) account for primary invagination,
adjacent cells having expanded apices, so suggesting that they there are no forces outside the immediate vicinity of the
are under tension (Hardin , 1989; 3.44), but interpretable vegetal plate required for its invagination, since the vegetal
experimental disruption of this process has been difficult (see plate can be isolated several hours before primary invagination
Hardin, 1990), Swelling pressure generated by secretion of begins and it will still invaginate on schedule (Moore and Burt,
proteoglycans into the lumen of the archenteron has also been 1939; Ettensohn, 1984),
suggested as a means by which the archenteron could in­ Considerably more is known about the elongation of the
vaginate (Morrill and Santos, 1985). In support of this idea, a archenteron and here epith elial cell rearrangement plays an

47
3.44 3.45

3.48

3.46 3.47

3.44-3.48 Formation of the archenteron LytechinU5 pictu5. 3.44 is a scanning electron micrograph of an early gastrula
(x1500). 3.45 and 3.46 (xSOO) show changes in shape of rhodamine-labelled clones during gastrulation (3.45, early gastrula;
3.46 , late gastrula ). 3.47 is an embryo is an embryo whose secondary mesenchyme cells have been ablated by a laser
microbeam. Elongation of the archenteron cea ses at two thirds of its normal final length (x500). 3.48 is an exogastrula
produced by treatment with lithium chloride (x500 ).

important role. Ettensohn (1985a) deduced that, as the arch­ invagination, the archenteron elongates to two-thirds of the
enteron elongates, the number of cells around the circum­ normal length, even though secondary mesenchyme cells do
ference of the archenteron decrea ses, while more direct not attach and pull in either case (Hardin and Cheng, 1986;
evidence for cell rearrangement comes from the behaviour of Hardin , 1988; 3.47, 3.48).
fluorescently labelled patches of cells within the vegetal plate The cellular processes which generate autonomous re­
during invagination: patches of such labelled cells gradually arrangement are not understood. Direct observation of cell
extend and narrow as cells interdigitate to lengthen the rearrangement in Eucidaris tribuloides suggests that cells
archenteron (3.45,3.46). Cell rearrangement appears to be the 'jostle ' against one another, and their basal ends display
dominant means by which the archenteron elongates, since vigorous motility (Hardin, 1989), but how such motility might
additional material is not added by mitosis (Stephens et al. , be translated into directed rearrangement is not known. In any
1986) or involution in L. pictus (Hardin, 1989). What forces ca se, completion of archenteron elongation requires the
drive this rearrangement? Several observations suggested that activity of secondary mesenchyme cells. In laser-irradiated
the filopodia of secondary mesenchyme cells can exert embryos in which a few filopodia are left intact, the arch­
significant tension and led to the hypothesis that filopodial enteron will continue to elongate after the two-thirds gastrula
traction causes the archenteron to elongate (Dan and Okazaki, stage, but more slowly than normal (Hardin, 1988). At about
1956; Gustafson, 1963 ; reviewed in Hardin , 1988), but two the time that secondary mesenchyme cells reach the animal
observations counter this suggestion. When filopodia are pole, there is a transient stretching of cells in the archenteron,
ablated with a laser microbeam or when embryos are induced apparently in response to filopodial traction (Hardin, 1989).
to exogastrulate, so producing an evagination rather than an

48
Cell interactions between secondary mesenchyme cells and the ectoderm

How do secondary mesenchyme cells aid the attachment of the the archenteron stably att aches ahead of schedule (3.51).
archenteron to a specific site in the ectoderm? When observed Second, when embryos are squeezed into narrow diameter
closely, the basic behaviour of secondary mesenchyme cells capillary tubing so that secondary mesenchyme cells cannot
involves continual extension of filopodia (3.49); these often attach to the animal pole, they continue their cyclical extension
remain attached for a time, but eventually detach and collapse, for a longer period of time than in normal embryos. [f the
only to be re-extended in a cyclical fashion (Gustafson and embryo is held in such a tube for several hours, some secondary
Kinnander, 1956; Hardin and McClay, 1990). Analysis of the mesenchyme cells eventually detach from the archenteron ,
duration of attachments of filopodia which make contact near migrate to the animal pole, and undergo the change in behaviour
the animal pole (3.50) indicate that they attach 20-50 times seen in normal embryos (3.52). [f the embryo is released from
longer than those making attachments at other sites (Hardin and the tube, it regains a spherical shape, and, as it does so, the tip of
McClay, 1990). Several lines of evidence indicate that second­ the archenteron rapidly attaches to the anim a l pole . Finally,
ary mesenchyme cells respond uniquely to this region (Hardin archenterons attach to the nearest avaiJable apical plate region in
and and McClay , 1990). First, when the animal pole region is fused mUltiple embryos . All of these experiments point to the
pushed towards the tip of the archenteron so that contact is existence of localized information in the animal pole region
allowed earli er than it would normall y occur, the cyclical which elicits this specific change in the motility and behaviour
behaviour of the secondary mesenchyme cells ceases early, and of secondary mesenchyme cells (Hardin and McClay, 1990).

3.49 3.50
3.49-3.52 Behaviour of secondary ... ... '. ,,'
.., . ..-"
,
..,..... """'C ....~."
:.
"
mesenchyme cells during gastrulation
in L. va riegatus. 3.49 (x7S0) shows a
I" >," " 3 '. . ~'V' •

r'· ,~::, :t~, '. l'

.
'-....::. ~,' (·\I;.~~.I. ~., ' ,. '.
laser scanning confocal image of a
" ~,'~;> w,' . ~.:... ':t'~,.\.3
midgastrul a stained with rhodamine­ ..... '~':\o~ • '~'~. ,i-:fY..
phalloidin . Filopodia radiate away '.c .•"'<'":.~
.. .. ..... ~ .•' ~\,) "~
''''
.• ,..".
~.
from the archenteron, producing a
' spray' of protrusions which make
contact with the ectoderm . 3.50
, .- \
~ .,:~;~}'
.• ·'.V·::·
,'.':J
.
~ ..
; '. :"',
..... ~.
'\
(x3S0) shows a late gastrula; sec­ "' :ft: .
-

, '- fl, .
~-.
ondary mesenchyme cells cease their
·r
".~ 'J
\t'., " , ••'f'o',~_-: ..',' • .1''.... ,,'.
. . ..
:,'....
;
protrusive activity when the y make
........ .,;
4 .1 "'~rl,
.. ~ ".~,

contact near the an i mal pole . 3.51

(x4S0) shows an embryo imprisoned
......... ':.~
. ~ .~

in nylon cloth; secondary mesen­

chyme cells attach prematu rely at the 3.51 3.52
animal pol e. 3.52 (xSOO ) shows an
embryo imprisoned in capillary tubing
for 2hr. Some secondary mesenchyme
cells have detached from the
archenteron and congregated at the
animal pole (arrow).

49
Phylogenetic differences in modes and timing of development

The sea urchin embryo has recently received attention as a after invagination of the archenteron has begun, even though
system for examining phylogenetic diversity during early these cells are derived from the micromeres (Wray and
development (Raff, 1987; Raff and Wray, 1989; Wray and McClay, 1987; Urben et al., 1988). Even more radical
Raff, 1990). In different sea urchin species, for example, there alterations are evident in direct-developing sea urchins where
seem to be several ways in which the archenteron can elongate, the production of a functioning larval gut does not occur and
all apparently accounted for by differences in embryonic shape whose metamorphosis is exceedingly rapid compared to sea
and placement of the future oral region (Hardin and McClay, urchins which pass through a true larval phase (3.53-3.56) .
1990). In addition to variations in embryonic shape, there are Here, features of early development which appear to be
also variations in the timing of developmental events with devoted to the production of exclusively larval structures are
respect to one another when different species are compared often completely lost (reviewed by Raff, 1987; Raff and Wray,
(heterochronies), with ingression of spiculogenic cells being a 1989; Wray and Raff, 1990). Other alterations include dramatic
good example of this sort of variation. Spicule-producing cells differences in cel1 lineages, mechanisms of cell fate
ingress at the mesenchyme blastula stage in the euechinoid sea determination along the dorsoventral axis, and mode of
urchin embryos with a typical larval mode of development, but gastrulation (reviewed in Raff, 1992» .
ingression of spiculogenic cells in Eucidaris occurs many hours

3.53 3.54
3.53-3.56 Direct develop­
ment of the Austral ian sea
urchin , Heliocidaris erythro­
gramma. 3.53 shows an un­
fertilized egg immersed in
orange ink to visualize the
jelly coat. 3.54 shows the
wrinkled gastrula stage. 3.55
shows a four-day-old embryo
immediately before meta­
morphosis, oral view. Tube
feet and vestibule are visible.
3.56 shows a juvenile shortly
after metamorphosis (xSO;
courtesy of L. Herlands).

3.55 3.56

50
The future
The sea urchin embryo occupies an important place in the altering the expression of specific genes, and of DNA binding
history of developmental biology, and continues to be a useful factors in the regulation of tissue-specific gene expression, a
experimental system today. As a model system for uniting particularly prominent feature of sea urchin development
experimental embryology and observation with the modern (reviewed by Davidson, 1989). As the tools of modern biology
tools of molecular and cell biology, the sea urchin embryo become increasingly refined and powerful, the simplicity of
continues to provide unique opportunities for studying early the sea urchin embryo and the ease with which it can be
development. Current work is focusing on the roles of cell manipulated will provide a rich context in which to study the
adhesion during gastrulation, of cell-cell interactions in molecular basis of early development.

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