1899
Development 124, 1899-1908 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV6282
Skeletal morphogenesis in the sea urchin embryo: regulation of primary
mesenchyme gene expression and skeletal rod growth by ectoderm-derived
cues
Kirsten A. Guss and Charles A. Ettensohn*
Department of Biological Sciences and Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, 4400
Fifth Avenue, Pittsburgh, PA 15213, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
The skeleton of the sea urchin embryo is synthesized by the
primary mesenchyme cells (PMCs). Previous studies have
shown that local interactions between PMCs and the neighboring ectoderm regulate several aspects of skeletal morphogenesis, including PMC distribution in the blastocoel,
the size of the skeleton and its branching pattern. In the
present study, we have further examined the regulation of
skeletogenesis by the ectoderm. We generated a ‘rate map’
of skeletal growth, which revealed stereotypical changes in
the rates at which specific skeletal elements elongate during
development. We showed that three transcripts encoding
PMC-specific gene products known to be involved in the
synthesis of the skeleton exhibited dynamic, spatially
regulated patterns of expression within the PMC
syncytium. All three gene products showed high levels of
expression at sites of skeletal rod growth, although the
specific patterns varied among the genes. We present direct
evidence, based upon cell transplantation experiments, that
the expression of one of these genes, SM30, is responsive to
local, ectoderm-derived cues. Based upon our studies, we
suggest that short-range signals from different ectodermal
territories may regulate the expression of PMC-specific
gene products that are rate-limiting in skeletal biosynthesis, thereby locally influencing skeletal rod growth.
INTRODUCTION
proteins (Sucov et al., 1987; Livingston et al., 1991; KatohFukui et al., 1991, 1992; Harkey et al., 1995), whereas SM30
is an acidic glycoprotein (George et al., 1991). The specific
roles of these matrix proteins have not yet been elucidated,
although they may function in the nucleation or orientation of
crystal growth (Wilt and Benson, 1988; Harkey et al., 1995).
Previous studies have shown that the ectoderm plays an
important role in PMC guidance, skeletal patterning and
skeletal rod elongation (Ettensohn, 1990; Hardin et al., 1992;
Armstrong et al., 1993; Ettensohn and Malinda, 1993;
Ettensohn et al., 1997). One means by which the ectoderm
might influence skeletal morphogenesis is by controlling the
expression of PMC-specific gene products involved in spicule
biosynthesis. Harkey et al. (1992) found that the msp130 gene
exhibits a complex pattern of spatial regulation within the PMC
syncytium during skeletogenesis. At ingression, msp130
mRNA is expressed at high levels by all PMCs, but later in
development this gene is down-regulated by PMCs that are
localized in specific regions of the developing skeletal system.
This suggests that at later developmental stages, the expression
of msp130 is regulated by extrinsic cues. In support of this
view, cell transplantation studies have shown that all PMCs
initially have the capacity to migrate to any region of the subequatorial ring pattern and, therefore to contribute to any part
of the developing skeletal system (Ettensohn, 1990). Molecular
The skeleton of the sea urchin embryo is synthesized by
primary mesenchyme cells (PMCs), descendants of the
micromeres of the 16-cell stage embryo (for reviews see
Gustafson and Wolpert, 1967; Okazaki, 1975a; Ettensohn et
al., 1997). During gastrulation, the PMCs gradually become
arranged in a characteristic ring pattern, composed of two
aggregates of cells (the ventrolateral clusters) connected by cell
chains extending circumferentially on the ventral and dorsal
surfaces of the blastocoel (the ventral and dorsal chains,
respectively). As the ring is forming, filopodial protrusions of
the PMCs fuse, linking the cells in a syncytial network (Hodor
and Ettensohn, 1995). Skeletogenesis begins with the
formation of one triradiate spicule rudiment in each of the two
ventrolateral clusters. Subsequently, the spicule rudiments
elongate and branch in a stereotyped fashion to give rise to the
embryonic skeleton.
Several PMC-specific gene products that participate in the
synthesis of the skeleton have been identified. Msp130 is a
sulfated cell-surface glycoprotein that has been implicated in
calcium uptake or deposition (Carson et al., 1985; Leaf et al.,
1987). Three proteins of the spicule matrix, SM50, SM30 and
PM27, have also been analyzed in detail. SM50 and PM27 are
predicted to be structurally similar, nonglycosylated, basic
Key words: sea urchin embryo, primary mesenchyme,
skeletogenesis, msp130, SM50, SM30, morphogenesis, cell
interactions, gene expression
1900 K. A. Guss and C. A. Ettensohn
differences among PMCs that arise after the formation of the
ring pattern must, be a consequence of different stimuli that act
on the cells.
In this study, we have further examined the regulation of
skeletogenesis by the ectoderm. We provide both cellular and
molecular evidence for local, ectodermal control of skeletal rod
growth. Our results suggest that the ectoderm may control
skeletal morphogenesis by regulating the expression of PMCspecific gene products involved in spicule biogenesis.
MATERIALS AND METHODS
Embryo culture
Adult L. variegatus were maintained and embryos cultured according
to Ettensohn and Malinda (1993). The rate of development was controlled by incubating the embryo cultures at 18-25°C in constant-temperature water baths.
Calcein labelling
Because the rate of skeletal rod elongation varies with temperature,
all embryos used for calcein labelling were cultured at 23°C. Calcein
was prepared as a 1.25 mg/ml stock solution in Instant Ocean (IO),
pH 8.3, and stored at 4°C. At selected stages, embryos were incubated
for 1 hour in IO containing 50 µg/ml calcein, then washed and transferred to fresh IO for 2 hours. Labelled embryos were then fixed in
4% paraformaldehyde in IO at room temperature (RT) for 2 hours,
followed by post-fixation in 100% methanol at −20°C for 20 minutes.
Embryos were examined with epifluorescence optics using a standard
fluorescein filter set. The length of the unlabelled spicule material at
the tip of each skeletal rod was measured with an ocular micrometer,
and this value was divided by 2 hours to give an average rate of rod
growth (in µm/hour) during the postlabelling period.
RNA isolation and northern blot analysis
Total RNA was isolated from embryos using the method of Chomczynski and Sacchi (1987), with an additional precipitation of RNA
with 8 M lithium chloride. Poly(A)+ RNA was isolated by subjecting
total RNA to affinity chromatography on oligo(dT)-cellulose
(Sambrook et al., 1989).
For northern blot analysis, total RNA (20-25 µg/stage) was
separated in 1% agarose gels containing 2.2 M formaldehyde and
transferred to nylon membranes (Sambrook et al., 1989). Blots were
hybridized overnight at 42°C in the presence of 50% formamide, with
[α-32P]dCTP-labelled DNA probes synthesized with the Boehringer
Mannheim random-primed DNA labelling kit. Blots were washed at
a final stringency of 0.2×SSC, 0.1% SDS at 42°C. Radioactive signal
was quantitated using an AMBIS 4000 radioanalytic imaging detector
(AMBIS, Inc).
L. variegatus homologues of SM50, SM30 and msp130
An oligo(dT)-primed L. variegatus mesenchyme blastula stage cDNA
library was constructed in λZAP (Stratagene) using poly(A)+ RNA.
To isolate a cDNA encoding the L. variegatus homologue of SM50,
this library was screened with LSM34, a cDNA that encodes the L.
pictus homologue of SM50 (Livingston et al., 1991). Filters were
hybridized overnight in the presence of 50% formamide at 42°C, and
washed at a final stringency of 0.1× SSC, 0.1% SDS at 65°C. A 0.9
kb cDNA was identified, and digested at an internal StuI site located
145 nucleotides from the 3′ end to eliminate poly(A)+ sequences. This
subclone, referred to as LvSM50-StuI, was used as template for
synthesis of DNA and RNA probes for L. variegatus SM50 (LvSM50)
transcripts.
A cDNA encoding the L. variegatus homologue of SM30 was
cloned by screening a L. variegatus prism stage cDNA library
(Wessel et al., 1989) with pNG7, a cDNA that encodes SM30 from S.
purpuratus (George et al., 1991). Hybridization was performed as
described above, and filters were washed at a final stringency of 0.2×
SSC, 0.1% SDS at 50°C. One 1.6 kb clone, LvSM30 (P2C), appeared
to be nearly full-length and was used for synthesis of DNA and RNA
probes for L. variegatus SM30 (LvSM30) transcripts.
Positive clones were excised in vivo as directed by Stratagene to
generate pBluescript SK(−) plasmids, and sequencing was performed
using Sequenase 2.0 (United States Biochemical). Nucleic acid
sequence similarity searches and alignments were performed using the
BLASTN program (Altschul et al., 1990) and MacVector (International Biotechnologies, Inc.).
A cDNA clone encoding a portion of the L. variegatus homologue
of msp130 was the gift of C. Marshall (UCLA) and R. Raff (Indiana
University), and was used to generate DNA and RNA probes for L.
variegatus msp130 (Lvmsp130) transcripts.
Whole-mount in situ hybridization
Whole-mount in situ hybridizations were performed as described by
Harkey et al. (1992) with the modifications of Ransick et al. (1993,
1995). To reduce background and extraneous binding of probes to
pigment cells (Fang and Brandhorst, 1996), some samples were
treated with 50 µg/ml ribonuclease A and 50 U/ml ribonuclease T1
following hybridization (Frudakis and Wilt, 1995). Digoxigenin-11labelled antisense and sense RNA probes were synthesized with the
MEGAscript in vitro transcription kit (Ambion) under the conditions
described by Ransick et al. (1993), and alkaline hydrolyzed. Bulk processing of fixed embryos was carried out entirely in 1.5 ml microfuge
tubes (approx. 50 µl packed embryos/tube). Small numbers of
embryos (fewer than 300) were fixed and processed in nylon meshbottomed baskets placed in flat-bottomed 24-well plates. For hybridization, embryos resuspended in hybridization buffer were drawn into
the narrow portion of a 9 inch pasteur pipette, which was then sealed
at both ends and submerged in a water bath. Embryos were returned
to the baskets for post-hybridization steps. Appropriate control
embryos were processed in parallel with microsurgically manipulated
embryos. Hybridizations with sense probes were performed for
Lvmsp130 and LvSM30 and showed no specific signal above background (not shown).
Embryo microsurgery
Depletions and transplantations of PMCs were carried out as
described by Ettensohn (1990). For heterochronic cell transplantations, PMCs isolated from mesenchyme blastula-stage donor embryos
were transplanted into the blastocoel of very early (pre-hatching)
blastula-stage hosts (20-70 PMCs/host). Host embryos were allowed
to develop for 5-5G hours at 24°C, and fixed for whole-mount in situ
hybridization. By this time, control embryos from the donor and host
batches had developed to the mid-late gastrula and mesenchyme
blastula stages, respectively. For isochronic (control) transplantations,
PMCs isolated from mesenchyme blastula-stage donor embryos were
transplanted into the blastocoel of same-stage recipients from which
the endogenous PMCs had been removed.
To remove triradiate spicule rudiments, mid gastrula-stage embryos
were immobilized in micromanipulation chambers (Ettensohn, 1990).
A micropipette with a continuous flow of seawater was used to flush
one of the two spicule rudiments and the associated PMCs out of the
blastocoel. Manipulated embryos were allowed to develop until
sibling embryos had reached the pluteus stage, when they were fixed
as described for calcein-labelled embryos (see above).
Nickel experiments
Embryos were ventralized with NiCl2 as described by Hardin et al.
(1992). Following fertilization, embryos were raised in 0.05-0.2 mM
NiCl2 in IO until the desired stage and fixed for in situ hybridization.
For cell transplantations, samples of sibling embryos were reared in
parallel in the presence and absence of 0.1 mM NiCl2 until the mesenchyme blastula stage. At this stage, the nickel-treated embryos were
Skeletal morphogenesis 1901
rinsed and transferred to IO, and approximately 50 PMCs from the
nickel-treated embryos were transplanted into untreated, sibling
embryos of the same stage from which the PMCs had been removed.
The embryos were allowed to develop to the late gastrula stage in IO,
when they were fixed for in situ hybridization.
RESULTS
Analysis of skeletal rod elongation in vivo
In a previous study, elongation rates of the postoral and body
rods were estimated by calculating changes in the mean lengths
of these rods at different time intervals (Ettensohn and
Malinda, 1993). Because this method involved comparing
averages of measurements made from populations of embryos,
we sought an alternative that would allow us to measure
directly elongation rates in individual embryos. A simple and
accurate method was to label spicules with calcein, a polyanionic derivative of fluorescein that binds Ca2+ and other
divalent cations (Wallach et al., 1959) (Fig. 1A,B).
Based upon the results of calcein-labelling experiments, we
constructed a ‘rate map’ of skeletal rod elongation (Fig. 1C).
This map revealed several unexpected features of skeletal rod
growth. The three arms of the triradiate spicule rudiments
initially elongated at the same rate — about 6 µm/hour at 23°C.
The three radii showed very different growth characteristics,
however, as development proceeded. The radius that gave rise
to the ventral transverse rod elongated for 8-10 hours (until the
late prism stage), when its growth ceased. The radius that
formed the dorso-ventral connecting rod extended toward the
animal pole at a constant rate of 6 µm/hour, then branched to
form the anterolateral and recurrent rods.
Initially, the anterolateral rod continued to
extend at about this same rate, although it
elongated more rapidly late in embryogenesis.
The third radius of the rudiment, the anonymous
rod, extended only a short distance before
branching to form the postoral and body rods.
After branching, these rods more than doubled
their rate of elongation. The body rod extended
Fig. 1. The calcein-labelling method and a map of
skeletal rod elongation rates. (A, B) Brightfield and
epifluorescence micrographs of one postoral arm of
an early pluteus larva that was incubated in calcein
for 1 hour, followed by a 2 hour ‘chase’ in the
absence of dye (see Materials and methods). Spicule
elongation after the removal of calcein has created an
unlabeled zone of material at the rod tip (indicated
by arrowheads). Scale bar, 15 µm. (C) A map of
skeletal rod elongation rates. The heavy black lines
represent the skeletal rods of a two-armed pluteus
larva. Numbers along the rods indicate the
elongation rates (in µm/hour) of the rods at different
stages of growth. Each value represents an
independent measurement determined by calcein
labelling, and data from six different batches of
embryos were pooled. The position of each value
(thin black lines) indicates the length of the rod at
the mid-point of the corresponding chase period.
Boxes show means and standard deviations of all
rate measurements for each individual rod.
rapidly to the posterior apex of the prism stage embryo,
slowing near the end of its growth as the branches of the
scheitel began to form. The postoral rod continued to elongate
rapidly throughout embryogenesis, reaching a maximum rate
of 14-15 µm/hour.
The ventral transverse rods were the only branches of the
skeleton that ceased to elongate during embryogenesis. The tips
of these rods eventually meet at the ventral midline, suggesting
that contact between the rods might restrict their growth. To test
this hypothesis, we eliminated one of the two triradiate spicule
rudiments (and the PMCs associated with it) at the mid-gastrula
stage and examined the elongation of the remaining ventral
transverse rod (Fig. 2). During the post-operative period, the
half-skeleton on the operated side usually re-formed, at least
partially. We excluded from our analysis embryos in which the
regenerated ventral transverse rod came into contact with the
ventral transverse rod on the unoperated side. In the remaining
embryos (n=20), in three cases no skeleton re-formed on the
operated side, and in the remaining 17 cases, the average separation between the tips of the ventral transverse rods at the late
pluteus stage was 20.8 µm (s.d.=10.8 µm).
In these 20 embryos, the average length of the ventral transverse rods on the unoperated side was 1.2× the length of ventral
transverse rods in control, sibling embryos (s.d.=0.2). The
maximum length of the ventral transverse rods in control
embryos is 55-60 µm; an additional 20% in length represents
11-12 µm of spicule. At an average growth rate of 5.6 µm/hour
(Fig. 1C), about 2 hours would be required to deposit this much
material. During normal development, however, other rods
(e.g. the postoral rod) continued to elongate for at least 10
hours after the ventral transverse rods ceased their growth.
1902 K. A. Guss and C. A. Ettensohn
Fig. 2. Microsurgical removal of one triradiate spicule rudiment.
(A) Ventral view of a late gastrula-stage embryo immediately after
microsurgery. One triradiate rudiment has been removed along with
the associated PMCs (arrowhead), leaving a single spicule rudiment
(arrow). Scale bar, 30 µm. (B) Pluteus larva derived from such an
embryo. A half-skeleton, including a ventral transverse rod
(arrowhead), has partially reformed on the operated side of the
embryo. The ventral transverse rod on the unoperated side of the
embryo (arrow) is only slightly longer than normal, even though the
tips of the two ventral transverse rods remain well separated. Scale
bar, 50 µm.
Even in the absence of its partner, therefore, the ventral transverse rod slowed or stopped its growth while other rods were
still elongating rapidly. We concluded that although contact
between the tips of the ventral transverse rods plays a limited
role in restricting their length, other factors cause these rods to
terminate at a relatively early stage of development.
Isolation of probes for molecular analysis: cloning
of LvSM50 and LvSM30
In order to carefully compare the temporal and spatial
expression patterns of several PMC markers in a species
amenable to embryo micromanipulation, homologues of SM30
and SM50 were obtained in L. variegatus (Materials and
Methods). Alignment of approx. 300 nucleotides of sequence
information obtained from the L. variegatus homologue of
SM50 with the L. pictus homologue (Livingston et al., 1991),
and approx. 250 nucleotides with the S. purpuratus (Sucov et
al., 1987; Katoh-Fukui et al., 1991) and H. pulcherrimus
(Katoh-Fukui et al., 1992) homologues, showed nucleic acid
sequence identities of approximately 90%, 70% and 70%,
respectively. Alignment of approx. 300 nucleotides from the L.
variegatus homologue of SM30 with the S. purpuratus
homologue (George et al., 1991) showed nucleic acid sequence
identities of approximately 70%. Based on these sequence similarities and the spatial and temporal expression patterns
described below, we concluded that these cDNAs corresponded to transcripts encoded by the L. variegatus SM50 and
SM30 genes, referred to as LvSM50 and LvSM30.
Expression of PMC-specific transcripts in L.
variegatus
By northern blot analysis, the Lvmsp130 probe recognized a
transcript of approx. 4 kb, which was detectable by the hatched
blastula stage (Fig. 3). Expression levels peaked at the mid
gastrula stage and decreased through the late pluteus stage. By
whole-mount in situ hybridization, expression was detectable
in the vegetal plate of hatched blastula-stage embryos (Fig.
4A), prior to PMC ingression. All PMCs in the embryo
Fig. 3. (A) Northern blot hybridization showing expression of
Lvmsp130, LvSM50 and LvSM30 transcripts during development. A
ubiquitin probe was used to determine the relative amounts of
hybridizable RNA in different lanes (Gong et al., 1991). The bottom
panel shows the 18S rRNA band on the ethidium bromide-stained
gel. Size standards were based on the migration of rRNAs. HB,
hatched blastula; MB, mesenchyme blastula; G, gastrula; PR, prism;
PL, pluteus larva. (B) Quantitation of radioactive signals from blots
shown in A. Values were corrected for relative amounts of
hybridizable RNA/lane based on ubiquitin expression levels.
Maximal values were normalized to developmental stages with the
highest levels of expression of a particular mRNA. Error bars
represent two standard deviations calculated based on counting
efficiency during quantitation.
appeared to express equal levels of Lvmsp130 through the late
gastrula stage (Figs 4C,D, 7). By the prism stage (Fig. 4E),
however, a gradual reduction in transcript level had begun in
Skeletal morphogenesis 1903
Fig. 4. Whole-mount in situ
hybridizations showing
Lvmsp130 transcript expression
during embryogenesis.
(A) Hatched blastula. Expression
is detectable in the vegetal plate
(arrow) prior to PMC ingression.
(B) Late mesenchyme blastula.
(C,D) Late gastrula (two focal
planes of one embryo). At early
stages of gastrulation, all PMCs
express similar levels of
Lvmsp130 mRNA, including
PMCs in the ventrolateral
clusters (arrow, C) and the
longitudinal (arrowhead, C) and
ventral chains (arrow, D).
(E,F) Prism stage. Highest
expression is exhibited by PMCs
in the ventrolateral clusters
(arrow, E), particularly those along the anonymous rods (arrow, F). (G,H) Early pluteus
(two focal planes of one embryo), showing high expression in PMCs at the tips of the
budding postoral arms (arrow, G). Other cells throughout the embryo exhibit reduced
expression, including those at the tips of the dorsoventral connecting rods (arrow, H).
(I,J) Late pluteus (two focal planes of one embryo). PMCs at the tips of the postoral
(arrow, I) and anterolateral rods (arrow, J) exhibit the highest transcript levels, with
intermediate expression in the scheitel (arrowhead, I). Expression is no longer detectable
in PMCs along the shafts of the skeletal rods.
PMCs throughout the embryo except those located in the ventrolateral clusters. By the pluteus larva stage (Fig. 4I,J), transcript levels were highest in cells at the tips of the elongating
anterolateral and postoral rods, while intermediate levels of
expression were visible in cells in the scheitel. Expression of
Lvmsp130 transcript was no longer detectable in cells distributed along the shafts of the skeletal rods. Therefore, the
decreased levels of expression at later stages apparent by RNA
blot analysis appeared to be due to a reduction of transcript
levels in only a subset of the PMCs.
The LvSM50 transcript was approx. 1.6 kb in length (Fig. 3).
Although very faint signal was detectable at the hatched
blastula stage by northern analysis, levels increased significantly at the early mesenchyme blastula stage. LvSM50 transcripts increased in abundance as development proceeded. By
whole-mount in situ hybridization, LvSM50 transcripts were
first detectable in PMCs at the mesenchyme blastula stage (Fig.
5B). Like Lvmsp130, this transcript was initially expressed at
approximately equal levels in all PMCs but showed reproducible, regional variations in expression at later developmental stages. Specifically, a distinct increase in abundance was
exhibited by PMCs in the scheitel by the late pluteus stage
(Figs 5E,F, 7).
The LvSM30 probe recognized a transcript of approx. 2 kb
Fig. 5. LvSM50 transcript expression during embryogenesis.
(A) Hatched blastula. LvSM50 mRNA is undetectable prior to PMC
ingression. (B) Late mesenchyme blastula. (C,D) Late gastrula (two
focal planes of one embryo). During most of skeletogenesis, similar
levels of LvSM50 transcript are exhibited by all PMCs, including
cells in the ventral chain (arrow, C) and ventrolateral clusters (arrow,
D). (E,F) Pluteus larva (two focal planes of one embryo) showing the
anal surface (E) and oral hood (F). PMCs in the scheitel express the
highest levels of the transcript (arrow, E).
that was first detectable at low levels at the mesenchyme
blastula stage (Fig. 3). Expression increased during gastrulation and through the late pluteus larva stage. By whole-mount
1904 K. A. Guss and C. A. Ettensohn
Fig. 6. LvSM30 transcript
expression during
embryogenesis. For each
developmental stage except
pluteus, a lateral view and a
view along the animal-vegetal
(A/V) axis are shown.
(A,B) Early gastrula. Expression
is initiated in cells that are
forming the ventrolateral
clusters (arrow, B). (C,D) Late
gastrula. PMCs in the
ventrolateral clusters (arrows)
express the highest levels of
transcript. Cells in the dorsal
chain (bold arrow, D), and
ventral chain (arrowhead, D)
express intermediate and
undetectable levels, respectively.
(E,F) Prism stage. High
expression is maintained by
PMCs in the ventrolateral
clusters (arrows), especially
those along the anonymous rods.
PMCs in the ventral chain and
near the tips of the longitudinal
chains (arrowhead, E) do not
express detectable levels of LvSM30. (G,H) Early pluteus (two focal planes of one embryo). High levels of expression are maintained by
cells at the tips of the postoral arms (arrow, G). Most PMCs except those along the ventral transverse rods (arrowhead, G) show increased
levels of expression. (I,J) Pluteus larvae showing the anal surface (I) and oral hood (J). High expression is exhibited by all PMCs except
those along the ventral transverse rods (arrow, I).
in situ hybridization, we observed that, unlike Lvmsp130 and
LvSM50, which appeared to initially accumulate equally in all
PMCs, LvSM30 expression commenced in a subset of the
PMCs. During early gastrulation (Figs 6A,B, 7), LvSM30 transcripts accumulated specifically in PMCs located in the ventrolateral clusters of the forming ring. By the late gastrula
stage, a striking pattern of expression in different regions of
the PMC ring was visible (Fig. 6C,D). Cells located in the ventrolateral clusters exhibited the highest level of LvSM30
expression, whereas cells along the dorsal branch of the ring
exhibited intermediate levels. Expression was undetectable in
cells in the ventral chain and at the tips of the longitudinal
chains that extend toward the animal pole from the ventrolateral clusters. At later stages, cells at the tips of the longitudinal chains began to express LvSM30 as they migrated toward
the animal pole (Fig. 6E,H). By the pluteus larva stage, strong
expression was exhibited by all PMCs in the embryo except
those located along the ventral transverse rods (Fig. 6I,J).
LvSM30 expression in embryos with altered
numbers of PMCs
To determine if the activation of LvSM30 expression in the ventrolateral clusters was a result of the aggregation of PMCs at
these sites, we examined the pattern of expression in embryos
in which the number of PMCs had been altered microsurgically (Ettensohn, 1990). In PMC-deficient embryos (containing 3-30 PMCs; n=30), the highest expression levels were seen
in the ventrolateral clusters, despite the reduced numbers of
cells at these sites (Fig. 8A). In some embryos with very few
PMCs, the positions of the ventrolateral clusters could not be
determined. Regardless, we observed LvSM30 expression in
SM30
SM50
msp130
Fig. 7. Summary of LvSM30, LvSM50 and Lvmsp130 transcript
distributions at the late gastrula (left) and late pluteus (right) stages,
based on whole-mount in situ hybridization data.
Skeletal morphogenesis 1905
Fig. 8. LvSM30 transcript expression in embryos
with altered numbers of PMCs. (A) A PMCdeficient embryo. LvSM30 is expressed in
‘clusters’ of as few as three PMCs (arrow).
(B,C) Two focal planes of one PMCsupplemented embryo. Highest expression is
still localized to cells of the ventrolateral
clusters (arrow, B). Despite the increased cell
density along the ventral chain (arrow, C), these
cells do not express detectable levels of LvSM30.
aggregates of as few as three cells. These observations indicate
that clustering of PMCs is not necessary for the activation of
LvSM30 expression, at least to moderate levels. In PMC-supplemented embryos (containing 2-3 times the normal complement of PMCs, n=7), despite the greatly increased density of
cells along the ventral chain, these cells did not express
detectable levels of LvSM30 (Fig. 8B,C). Therefore, high cell
density is not sufficient to induce LvSM30 expression.
LvSM30 expression in embryos with ventralized
ectoderm
To test whether the spatial pattern of LvSM30 expression was
regulated by ectoderm-derived signals, we examined the
expression of this gene in ventralized embryos. NiCl2 ventralizes the ectoderm; as a consequence, the arrangement of PMCs
in the blastocoel is altered and the number of spicule rudiments
is increased (Hardin et al., 1992; Armstrong et al., 1993).
When embryos were raised continuously in NiCl2, both the
patterning of the PMC ring and the expression of LvSM30 were
altered. In most NiCl2-treated embryos, LvSM30 expression
was relatively uniform throughout the PMC ring (Fig. 9A). In
some embryos, multiple, discrete clusters of cells expressing
high levels of LvSM30 were visible, with lower transcript levels
exhibited by cells connecting these clusters (Fig. 9B). This
pattern of expression was more common in embryos cultured
in higher concentrations of nickel (e.g. 0.2 mM; not shown).
To determine whether the aberrant LvSM30 expression
pattern was due to an effect of nickel on the ectoderm or the
PMCs, we examined LvSM30 expression after transplanting
nickel-treated PMCs into untreated, PMCdepleted host embryos. In such embryos,
nickel-treated PMCs formed a ring and
exhibited a pattern of LvSM30 expression
Fig. 9. Misregulation of LvSM30 expression in
ventralized embryos. (A,B) LvSM30
expression in embryos treated continuously
from fertilization with 0.05 mM NiCl2, viewed
along the A/V axis (A) and laterally (B). In
most embryos, PMCs throughout the ring
exhibit high levels of LvSM30 expression
(arrow, A). In some cases, multiple clusters of
PMCs expressing high levels of LvSM30
(arrow, B) are connected by chains of cells
expressing lower levels. (C-E) Expression of
LvSM30 following transplantation of nickeltreated PMCs into an untreated, PMCdeficient host (three focal planes of a single
embryo). LvSM30 expression is highest in the
two ventrolateral clusters (arrows, C,D) and
lower in the dorsal chain (arrow, E).
similar to that observed in control embryos (Fig. 9C-E). In 6/7
cases, the PMCs formed a ring in which two clusters and either
the ventral and/or the dorsal chain were distinguishable. PMCs
in the two ventrolateral clusters (Fig. 9C,D) exhibited the
highest expression of LvSM30. The dorsal chain (Fig. 9E),
when present, exhibited faint to strong staining, whereas the
ventral chain showed consistently low expression. As a control,
a sample of nickel-treated donor embryos was allowed to
continue development in IO in parallel with the experimental
embryos. These donor embryos were ventralized and showed
an aberrant pattern of LvSM30 expression, as described above.
These results indicate that nickel causes a misregulation of
LvSM30 expression by altering the ectoderm.
Heterochronic cell transplantations
Transplantation of PMCs into recipient embryos of different
developmental stages has been used to provide evidence that
aspects of PMC behavior are regulated by extrinsic cues
(Ettensohn and McClay, 1986). We transferred PMCs from
mesenchyme blastulae into younger embryos (pre-hatching
blastulae) and examined LvSM30 expression after 5-5G hours,
when sibling donor embryos had reached the mid-late gastrula
stage and were expressing high levels of LvSM30 mRNA. As
a control for the transplantation procedure, PMCs were
removed from mesenchyme blastulae and microinjected into
recipient embryos of the same developmental stage from which
all endogenous PMCs had been removed (isochronic controls).
In the combined results of three series of heterochronic
transplants, in 23 of 40 experimental embryos, the PMCs
1906 K. A. Guss and C. A. Ettensohn
Table 1. Expression of LvSM30 by transplanted PMCs
Heterochronic transplants
Triala
1
2
3
4
Time
(hours)b
5.5
5.25
5.25
−
Isochronic transplants
No. of positive cells (cases)
0
1-2
>2
Time
(hours)b
5/9
12/13
6/18
−
0/9
1/13
5/18
−
4/9
0/13
7/18
−
−
5
5.25
5.5
aEach trial denotes a set of experiments performed with a single batch of embryos.
bPeriod of incubation at 24°C following cell transplantation.
cCases in which a PMC ring was clearly visible were additionally scored for the appearance
No. of positive cells (cases)
0
>2
−
1/14
0/6
0/7
−
13/14
6/6
7/7
Spatial regulation
(cases)c
−
7/8
6/6
4/4
of some degree of spatial regulation of LvSM30 expression (e.g.
the highest level of expression was localized to at least one cluster).
showed no significant signal above background (Table 1, Fig.
10A). The remaining embryos contained 1-2 cells exhibiting
signal above background (6/40) or more than two cells above
background (11/40). We differentiated between these last two
classes because the positively staining cells in the latter were
clearly identifiable as PMCs and were usually in clumps,
whereas the former were isolated and their identification as
PMCs was ambiguous. Sibling donor embryos fixed at the
same time exhibited a well-formed ring of PMCs that
expressed high levels of LvSM30 (not shown). Sibling host
embryos had reached the mesenchyme blastula stage, with
PMCs that were not yet expressing detectable levels of
LvSM30 expression (not shown).
In contrast to the low levels of LvSM30 expression observed
following heterochronic transplantations, 26/27 isochronic
control embryos exhibited more than two positive cells,
although signal intensity ranged from just above background
to very strong. In 17 of those embryos, a ring was distinguishable and the PMCs exhibited some degree of spatial regulation of LvSM30 expression, e.g. the highest degree of signal
was localized to cells in at least one cluster (Fig. 10B). In the
remaining cases, the presence of a ring could not be confirmed
because of the orientation of the embryos (five cases), or the
ring was incompletely formed and/or there were no differences
in levels of staining in cells in different regions of the ring (four
cases). We conclude that the delay in LvSM30 expression
following heterochronic PMC transplantation cannot be attrib-
Fig. 10. Heterochronic transplants. Expression of LvSM30 exhibited
by PMCs in representative cases of embryos, classified in Table 1 as
heterochronic transplants showing no positive cells (A) and
isochronic transplants showing more than two positive cells (B).
(A) In the majority of cases of heterochronic transplants, the cells did
not exhibit any signal above background. (B) In nearly every case,
isochronically transplanted PMCs were expressing LvSM30,
frequently in a spatially regulated manner similar to their
unperturbed late gastrula-stage siblings.
uted to the effects of microsurgery alone, since almost all of
the isochronic controls showed detectable levels of LvSM30
expression after the same period of time.
DISCUSSION
Local regulation of skeletal rod elongation rates and
transcript expression patterns
Our measurements using the calcein-labelling method show
that rates of skeletal rod elongation are more tightly regulated
during embryogenesis than was previously appreciated (e.g.
Ettensohn and Malinda, 1993). At all stages of skeletogenesis,
rates of spicule growth varied considerably in different regions
of the PMC syncytium. These variations in growth rate are not
simply attributable to differences in the numbers of PMCs
associated with the various skeletal rods or to changes in PMC
distribution during development (see Ettensohn, 1990;
Ettensohn and Malinda, 1993). An alternative hypothesis is
that growth rates reflect differences in the biosynthetic capacities of individual PMCs in different regions of the embryo.
Consistent with this view, we found that three PMC-specific
transcripts encoding gene products involved in skeletal
synthesis showed dynamic, spatially regulated patterns of
expression within the PMC syncytium.
We have described for the first time the spatial patterns of
expression of the SM50 and SM30 mRNAs. Like msp130, these
transcripts are expressed at the highest levels in regions of
active skeletal rod growth, i.e. at the sites of the triradiate
rudiments, the tips of the arm rods and/or the scheitel. When
the LvSM30 expression pattern is compared with the map of
skeletal rod growth, at least three additional correlations can
be made. First, the initial formation of the spicule primordia
within the two ventrolateral clusters is correlated with high
levels of LvSM30 expression at these sites. Second, the lowest
levels of LvSM30 mRNA expression are associated with the
PMCs located along the ventral aspect of the subequatorial
ring. Calcein labelling demonstrates that the ventral transverse
rod, which extends along this chain of PMCs, is the only
branch of the original triradiate spicule rudiment that ceases to
elongate during embryogenesis. By removing one of the two
triradiate spicule rudiments we have ruled out the possibility
that the primary cause of this growth arrest is contact between
the tips of the ventral transverse rods. Instead, it appears that
the blastocoel wall at the ventral midline provides a relatively
unfavorable local environment for spiculogenesis. Third, there
is a substantial increase in the rate of elongation of the postoral
Skeletal morphogenesis 1907
rod soon after it branches from the anonymous rod. At the same
time, PMCs in this region of the syncytium show elevated
levels of LvSM30 (as well as Lvmsp130) mRNA expression
(Figs 4E,F, 6E).
It is not known whether the expression patterns of the
proteins encoded by the LvSM30, LvSM50 and Lvmsp130
genes match those of the corresponding transcripts. Harkey et
al. (1995) reported that in S. purpuratus, the msp130 protein
is distributed evenly over the surfaces of all PMCs and spicules,
in apparent contrast to the spatially regulated expression of the
transcript (Harkey et al., 1992). It has recently been shown,
however, that in S. purpuratus the SM30 protein is expressed
at a reduced level along the ventral transverse rods, consistent
with the pattern of mRNA expression we have described in L.
variegatus (L. Urry and F. Wilt, personal communication). The
PM27 and SM50 proteins also have nonuniform distributions
in S. purpuratus, with relatively high levels of expression at the
spicule tips (Harkey et al., 1995).
Non-uniform distributions of the LvSM30, LvSM50 and
Lvmsp130 transcripts are established after the formation of the
PMC syncytium (Hodor and Ettensohn, 1995). The patterns of
mRNA localization we observed might, therefore, result from
directional transport or trapping of mRNAs within the
syncytium, in addition to possible local differences in rates of
mRNA degradation and/or synthesis (see review by St
Johnston, 1995). Our data indicate that the molecular
mechanism responsible for the heterogeneous distribution of
transcripts must be regulated by local cellular interactions.
Regulation of LvSM30 expression
Several features of LvSM30 expression suggest that this gene
product may play an especially important role in skeletal morphogenesis. SM30 is the most abundant spicule matrix protein
(F. Wilt, personal communication). As noted above, we found
a close correlation between local levels of LvSM30 expression
and skeletal rod growth in vivo. Other recent studies have
shown that SM30 expression is also tightly coupled to spicule
growth in vitro. In micromere cultures, cells that engage in
overt synthesis of spicules express SM30 transcript and protein,
while those cells that do not express this gene at detectable
levels do not secrete spicule material (F. Wilt, personal communication). In contrast, SM50 is expressed by all cells in the
same cultures.
Several lines of evidence suggest that the SM30 gene is
regulated differently than SM50 and msp130. In both L. variegatus (this study) and S. purpuratus (George et al., 1991),
SM30 mRNA accumulates distinctly later in development than
the other two transcripts. Moreover, from the earliest stage that
SM30 transcript can be detected by in situ hybridization, the
gene appears to be expressed nonuniformly within the PMC
population. In contrast, msp130 and SM50 transcripts are
initially expressed at approximately equal levels in all PMCs,
and show local modulations in expression only at later developmental stages. Lastly, SM30 expression in whole embryos is
sensitive to treatment with β-aminopropionitrile (BAPN), an
inhibitor of the post-translational processing of collagen, while
SM50 expression is unaffected by this drug (Stebbins, 1994).
This observation suggests that SM30 expression is regulated by
extracellular matrix (ECM)-mediated cues. Recent studies
using cultured micromeres have shown that expression of
SM30, but not other PMC-specific markers, is highly sensitive
to the presence of serum in the medium (X. Zhu and C. A.
Ettensohn, unpublished observations). The cell transplantation
experiments described in the present study provide direct
evidence that short-range, ectoderm-derived cues regulate
LvSM30 expression.
The regulation of skeletal morphogenesis
The morphogenesis of the skeletal system may be viewed as
an interplay between intrinsic, developmentally programmed
properties of the PMCs and ectoderm-derived signals that
regulate the molecular properties and behavior of the cells
(Ettensohn et al., 1996). We propose that the early determinative processes that rigidly direct the progeny of the micromeres
into a skeletogenic pathway of differentiation activate a basal,
autonomous program of gene expression in this lineage. The
basal program includes the initial activation of msp130 and
SM50, genes encoding proteins involved in cell-cell fusion, and
presumably other genes as well (Harkey et al., 1988; Stebbins,
1994; Hodor and Ettensohn, 1995). We suggest that the tight
regulation of skeletal morphogenesis observed in vivo is
achieved by coupling the expression of other genes, such as
SM30, to extrinsic cues. A requirement of this model is that at
least some of these signal-dependent genes encode proteins
that are rate-limiting for skeletogenesis. The local, ectodermderived signals may be mediated by the ECM and may involve
stimulatory factors analogous to those present in serum, EHS
matrix or blastocoel fluid (Okazaki, 1975b; Benson and
Chuppa, 1990; Kiyomoto and Tsukahara, 1991; Dolo et al.,
1992; Page and Benson, 1992). Later in embryogenesis, even
those genes that are autonomously up-regulated by PMCs as
part of the basal program come under local ectodermal influences, as indicated by the finding that all three PMC-specific
markers show spatially regulated expression at later stages.
According to this model, an important function of the basal,
autonomous developmental program of the micromeres is to
render the progeny of these cells receptive to external stimuli
that confront them at later developmental stages. The layering
of a program of signal-dependent gene activity onto an
autonomous, lineage-based developmental program provides a
means of coordinating the morphogenesis of the skeletal
system with that of other tissues in the embryo.
We thank R. Miller for technical assistance, and members of the
Pittsburgh Center for Marine Aquaculture for suggestions throughout
the course of this work. We are grateful to C. Marshall, R. Raff, G.
Wessel, and F. Wilt for providing molecular probes, T. Oligino for
sequencing, G. Wray for suggesting calcein labelling as a method to
study spicule growth, and T. Andacht, G. Cannon and D. McClay for
southern hospitality at DUML. This research was funded by NIH
Grant HD-24690 and an NIH Research Career Development Award,
both to C. A. E.
REFERENCES
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990).
Basic local alignment search tool. J. Mol. Biol. 215, 403-410.
Armstrong, N., Hardin, J. and McClay, D. R. (1993). Cell-cell interactions
regulate skeleton formation in the sea urchin embryo. Development 119, 833840.
Benson, S., Sucov, H., Stephens, L., Davidson, E. and Wilt, F. (1987). A
lineage-specific gene encoding a major matrix protein of the sea urchin
embryo spicule. I. Authentication of the cloned gene and its developmental
expression. Dev. Biol. 120, 499-506.
1908 K. A. Guss and C. A. Ettensohn
Benson, S. and Chuppa, S. (1990). Differentiation in vitro of sea urchin
micromeres on extracellular matrix in the absence of serum. J. Exp. Zool.
256, 222-226.
Carson, D. D., Farach, M. C., Earles, D. S., Decker, G. L. and Lennarz, W.
J. (1985). A monoclonal antibody inhibits calcium accumulation and
skeleton formation in cultured embryonic cells of the sea urchin. Cell 41,
639-648.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.
Biochem. 162, 156-159.
Dolo, V., Forti, C., Dell’Utri, S., Ghersi, G. and Vittorelli, M. L. (1992). An
acid extract from dissociation medium of sea urchin embryos, induces
mesenchyme differentiation. Cell Biol. Int. Rep. 16, 517-532.
Ettensohn, C. A. and McClay, D. R. (1986). The regulation of primary
mesenchyme cell migration in the sea urchin embryo: transplantations of
cells and latex beads. Dev. Biol. 117, 380-391.
Ettensohn, C. A. (1990). The regulation of primary mesenchyme cell
patterning. Dev. Biol. 140, 261-271.
Ettensohn, C. A. and Malinda, K. M. (1993). Size regulation and
morphogenesis: a cellular analysis of skeletogenesis in the sea urchin
embryo. Development 119, 155-167.
Ettensohn, C. A., Guss, K. A., Malinda, K. A., Miller, R. N., and Ruffins, S.
W. (1996). Cell interactions in the sea urchin embryo. In Advances in
Developmental Biochemistry, Volume VI (ed. P. Wasserman). pp. 47-98. JAI
Press, Greenwich.
Ettensohn, C. A., Guss, K. A., Hodor, P. G., and Malinda, K. M. (1997). The
morphogenesis of the skeletal system of the sea urchin embryo. In Progress
in Developmental Biology,Volume VIII, Reproductive Biology of
Invertebrates (ed. J. Collier). New York: Wiley and Sons, in press.
Fang, H., and Brandhorst, B. (1996). Expression of the actin gene family in
embryos of the sea urchin Lytechinus pictus. Dev. Biol. 173, 306-317.
Frudakis, T. N., and Wilt, F. (1995). Two cis elements collaborate to spatially
repress transcription from a sea urchin promoter. Dev. Biol. 172, 230-241.
George, N. C., Killian, C. E. and Wilt, F. H. (1991). Characterization and
expression of a gene encoding a 30.6-kDa Strongylocentrotus purpuratus
spicule matrix protein. Dev. Biol. 147, 334-342.
Gong, Z., Cserjesi, P., Wessel, G. M. and Brandhorst, B. P. (1991). Structure
and expression of the polyubiquitin gene in sea urchin embryos. Mol. Reprod.
Dev. 28, 111-118.
Gustafson, T. and Wolpert, L. (1967). Cellular movement and contact in sea
urchin morphogenesis. Biol. Rev. 42, 442-498.
Hardin, J., Coffman, J. A., Black, S. D. and McClay, D. R. (1992).
Commitment along the dorsoventral axis of the sea urchin embryo is altered
in response to NiCl2. Development 116, 671-685.
Harkey, M. A., Whiteley, H. R. and Whiteley, A. H. (1988). Coordinate
accumulation of five transcripts in the primary mesenchyme during
skeletogenesis in the sea urchin embryo. Dev. Biol. 125, 381-395.
Harkey, M. A., Whiteley, H. R. and Whiteley, A. H. (1992). Differential
expression of the msp130 gene among skeletal lineage cells in the sea urchin
embryo: a three dimensional in situ hybridization analysis. Mech. Dev. 37,
173-184.
Harkey, M. A., Kleug, K., Sheppard, P. and Raff, R. A. (1995). Structure,
expression, and extracellular targeting of PM27, a skeletal protein associated
specifically with growth of the sea urchin larval spicule. Dev. Biol. 168, 549566.
Hodor, P. H. and Ettensohn, C. A. (1995). Cell-cell fusion in the sea urchin
primary mesenchyme. Dev. Biol. 170, 745a.
Katoh-Fukui, Y., Noce, T., Ueda, T., Fujiwara, Y., Hashimoto, N.,
Higashinakagawa, T., Killian, C. E., Livingston, B. T., Wilt, F. H.,
Benson, S. C., Sucov, H. M. and Davidson, E. H. (1991). The corrected
structure of the SM50 spicule matrix protein of Strongylocentrotus
purpuratus. Dev. Biol. 145, 201-202.
Katoh-Fukui, Y., Noce, T., Ueda, T., Fujiwara, Y., Hashimoto, N., Tanaka,
S. and Higashinakagawa, T. (1992). Isolation and characterization of
cDNA encoding a spicule matrix protein in Hemicentrotus pulcherrimus
micromeres. Int. J. Dev. Biol. 36, 353-361.
Kiyomoto, M. and Tsukahara, J. (1991). Spicule formation-inducing
substance in sea urchin embryo. Dev. Growth Diff. 33, 443-450.
Leaf, D. S., Anstrom, J. A., Chin, J. E., Harkey, M. A., Showman, R. M. and
Raff, R. A. (1987). Antibodies to a fusion protein identify a cDNA clone
encoding msp130, a primary mesenchyme-specific cell surface protein of the
sea urchin embryo. Dev. Biol. 121, 29-40.
Livingston, B. T., Shaw, R., Bailey, A. and Wilt, F. (1991). Characterization
of a cDNA encoding a protein involved in formation of the skeleton during
development of the sea urchin Lytechinus pictus. Dev. Biol. 148, 473-480.
Okazaki, K. (1975a). Normal development to metamorphosis. In The Sea
Urchin Embryo: Biochemistry and Morphogenesis (ed. G. Czihak), pp. 177232. New York: Springer-Verlag.
Okazaki, K. (1975b). Spicule formation by isolated micromeres of the sea
urchin embryo. Am. Zool. 15, 567-581.
Page, L. and Benson, S. (1992). Analysis of competence in cultured sea urchin
micromeres. Exp. Cell Res. 203, 305-311.
Ransick, A., Ernst, S., Britten, R. J. and Davidson, E. H. (1993). Wholemount in situ hybridization shows Endo 16 to be a marker for the vegetal
plate territory in sea urchin embryos. Mech. Dev. 42, 117-124.
Ransick, A. and Davidson, E. H. (1995). Micromeres are required for normal
vegetal plate specification in sea urchin embryos. Development 121, 32153222.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory.
Stebbins, B. J. (1994). Disruptions of the extracellular matrix and expression
of spicule matrix genes. PhD Thesis, University of California, Berkeley, CA.
St Johnson (1995). The intracellular localization of mRNAs. Cell 81, 161-170.
Sucov, H. M., Benson, S., Robinson, J. J., Britten, R. J., Wilt, F. and
Davidson, E. H. (1987). A lineage-specific gene encoding a major matrix
protein of the sea urchin embryo spicule. II. Structure of the gene and derived
sequence of the protein. Dev. Biol. 120, 507-519.
Wallach, D. F., Surgenor, D. M., Soderberg, J., and Delano, E. (1959).
Preparation and properties of 3, 6-dihydroxy-2, 4-bis-[N,N′-di(carboxymethyl)-aminomethyl] fluoran. Anal. Chem. 31, 456-460.
Wessel, G. M., Goldberg, L., Lennarz, W. J. and Klein, W. H. (1989).
Gastrulation in the sea urchin is accompanied by the accumulation of an
endoderm-specific mRNA. Dev. Biol. 136, 526-536.
Wilt, F. H. and, S. C. (1988). Development of the endoskeletal spicule of the
sea urchin embryo. In Self-Assembling Architecture (ed. J. E. Varner), pp.
203-227. New York: Alan R. Liss.
(Accepted 7 March 1997)