Download Altered Cell Fate in LiCI-Treated Sea Urchin Embryos

DEVELOPMENTAL
BIOLOGY
147,445-450 (1991)
Altered Cell Fate in LiCI-Treated
CATHERINE
NOCENTE-MCGRATH~
ROBERT
Sea Urchin Embryos
MCISAAC,
Department of Biology, TV& University, Me#d,
AND SUSAN G. ERNSI?
Massachusetts 02155
Accepted July 5, 1991
End016 is a lineage-specific protein expressed during embryogenesis in the sea urchin, Strongykxentrotus purpuratus.
We have examined the effects of various concentrations of lithium chloride, a well-known vegetalizing agent, on End016
expression in whole sea urchin embryos. Our results show that treatment with LiCl causes increased steady-state levels
of End016 transcripts. An increase in the number of endodermal cells in treated embryos demonstrates a change in cell
lineage. In addition, we observed a delay in development of lithium-treated
embryos that is accompanied by a delay in
the expression of a late class histone H2B gene. o 1~1 Academic press, I~C.
INTRODUCTION
The profound effects of lithium
on embryogenesis
have intrigued developmental
biologists since its vegetalizing action on the sea urchin was first reported
nearly a century ago (Herbst, 1892). Lithium is thought
to affect cell fate determinations
in sea urchin (reviewed in Lallier, 1964) and frog embryos (Kao et al.,
1986), as well as Llictgostelium development (Peters et
aL, 1989). Renewed interest in this subject has been
sparked by the availability
of sensitive lineage-specific
probes and new information
on the physiological effects
of lithium. Recent studies have shown that lithium has
specific inhibitory effects on inositol phosphate metabolism. This has led to the hypothesis that lithium affects
embryogenesis by disrupting inductive interactions mediated by second messenger pathways (reviewed in
Berridge et aZ.,1989).
In the sea urchin embryo, lithium causes an exaggeration of vegetally derived tissues in whole embryos (reviewed in Lallier, 1964; Horstadius, 1973), and the appearance of vegetally derived structures in isolated animal caps (Livingston
and Wilt, 1989). The extent of
vegetalization
and the final morphology
depend upon
the concentration of lithium used, the time of exposure,
and the species of sea urchin. Classical studies on the
effects of lithium were limited by their reliance on morphological
criteria to identify differentiated
tissues.
The more recent studies by Livingston and Wilt (1989)
have employed molecular markers to demonstrate that
exposure to lithium can evoke the expression of vegetalspecific molecules in isolated animal blastomeres.
’ Present address: Department of Cellular and Developmental
ogy, Harvard University, Cambridge, MA 02138.
* To whom correspondence should be addressed.
Biol-
445
End016 is a vegetal-specific marker that is expressed
exclusively in endodermal tissue (Nocente-McGrath
et
aZ., 1989). The expression of this molecule can be detected in presumptive endoderm prior to the onset of
gastrulation.
After gastrulation,
expression of End016
becomes progressively restricted to a subset of endoderm tissue. The present study examines the effects of
lithium exposure on the temporal and spatial expression
of End016 using quantitative
hybridization
analysis and
indirect immunofluorescence
on whole mount and single cell preparations. We have also investigated the developmental delay observed in vegetalized embryos using a late histone H2B probe as a molecular index of
developmental
progression.
MATERIALS
AND METHODS
Animals. Adult Strongylocentrotw
pwpuratus were
purchased from Marinus, Inc. (Long Beach, CA). Maintenance of animals, spawning, fertilization,
and embryo
culture were as previously described (Pittman
and
Ernst, 1984). Dissociation of mesenchyme blastula embryos to single cells was accomplished using standard
methods (Pittman and Ernst, 1984). Embryos were pelleted, washed twice with calcium- and magnesium-free
sea water (CMFSW), and resuspended in 1 M glycine,
pH 7.8. Cell clumps were dispersed by gentle pipetting
and the cells were washed once with CMFSW prior to
application onto polylysine coated slides.
Lithium chloride vegetalization of sea urchin embryos.
Embryos were exposed to lithium chloride (LiCl) continuously between 30 min and 16 hr postfertilization.
Briefly, a l-liter culture of fertilized eggs was divided
equally into a control and three LiCl-treated
cultures.
This was accomplished by transferring
250 ml of embryos to three separate jars containing 750 ml of Milli0012-1666/91$3.00
Copyright
8 1991 by Academic Press. Inc.
All righta of reproduction
in any form reserved.
446
DEVELOPMENTAL
BIOLOGY
pore-filtered
sea water (MPFSW) supplemented
with
LiCl to final concentrations
of 15, 30, and 60 mM. The
control embryos (250 ml) were diluted to 1 liter and the
cultures were allowed to develop. At 16 hr postfertilization, embryos were pelleted by spinning 5 min at 400
rpm in a Damon CRU-5000 centrifuge. The pellets were
washed twice with MPFSW and resuspended in 1 liter of
MPFSW to continue development.
Northern blot analysis. Standard molecular biology
techniques were employed (Maniatis et a& 1982). Embryonic RNA was purified by the guanidine/CsCl
method
(Chirgwin et al, 1979), size fractionated on a 1% formaldehyde-agarose gel, and blotted to nitrocellulose.
The
filter was hybridized sequentially to =P-labeled probes
specific for End016 and for the late class histone H2B
gene (Maxson et al, 198313). Hybridization
conditions for
both clones were 40% formamide,
5~ SSCP, 5~ Denhardt’s, 0.5% SDS at 42°C. Filters were washed in 0.2X
SSC, 0.5% SDS at 65°C. Autoradiography
was performed with Kodak XAR5 film and a DuPont Cronex
intensifying
screen at -70°C.
Indirect immuno$uwescence. Whole mount embryos
and single cells were fixed and stained as previously
described (Nocente-McGrath
et al, 1989), with the following modification. Affinity-purified
anti-End016 antibodies were used at a 1:lOO dilution in 3% BSA/PBS. A
complete description of the affinity purification will appear elsewhere (manuscript
in preparation).
Briefly,
anti-End016
antibodies were purified by column chromatography over agarose coupled to a P-galactosidaseEnd016 fusion protein. Stained slides were mounted in
FITC-Guard
mounting medium (Testog Inc., Chicago,
IL). The reaction was viewed and photographed under a
Zeiss microscope equipped for epifluorescence using Kodak T-Max film at an ASA setting of 800. In addition,
dissociated preparations were stained for End016 and
counterstained with DAPI to determine the total number of cells in an individual field. The cells were photographed for End016 and for DAPI and three fields of
approximately
400 cells each were counted for End016
staining and DAPI staining.
RESULTS
Morphological Characteristics of Vegetalized Embyos
As a prerequisite for examining
the effects of LiCl
exposure on End016 expression, the overt morphological
characteristics
of S. pwpuratus embryos with varying
degrees of vegetalization
were documented.
Pluteus
stage embryos that had been cultured in the presence of
increasing amounts of lithium chloride were fixed and
photographed. Variation in the response to LiCl treatment by batches of eggs from different females was observed. The morphological
profile described below was
VOLUME
147,1991
obtained in all cultures, but the specific concentration of
lithium
required to produce the effects fluctuated
among batches of eggs collected from different females.
Untreated pluteus embryos (Fig. 1A) possess a welldeveloped three-part gut, a complete skeleton, and pigment cells distributed
throughout
the ectoderm. Embryos treated with 15 mMLiC1 (Fig. 1B) display everted
guts due to exogastrulation.
These embryos appear
nearly normal with respect to the proportions of the
primary tissue layers. In contrast, embryos cultured in
the presence of 30 mM LiCl exhibit a striking vegetalized morphology (Fig. 1C). These embryos appear as a
three-part “snowman” shape. The largest sphere is decorated with pigment cells and encloses skeletal spicules,
features characteristic
of larval ectoderm. The spicules
are small and abnormally
shaped. The two smaller
spheres are attached to the larger one by a ring of constricted cells and together appear to be a greatly enlarged gut. The smallest sphere often contains an invaginated structure. When embryos are exposed to 60 mM
LiCl, they undergo cleavage which results in relatively
normal blastulae, but they become increasingly
disorganized as morphogenesis
proceeds (data not shown).
They appear as variously sized masses of cells, small
clumps, and single cells by the time controls have completed embryogenesis. Pigment cells can be identified in
the masses, but there is little evidence of any tissue organization. These results are in agreement with earlier
reports that there is a graded response to increasing
LiCl concentrations
characterized by an increase in endoderm-like
tissue and a concurrent decrease in ectoderm.
Immurwlocalization of End016 in whole and Dissociated
Vegetalized Embryos
To investigate the spatial expression of End016 in vegetalized embryos, whole, fixed embryos were stained
with affinity-purified
anti-End016
antibodies (Fig. 2).
Embryo cultures were exposed to various concentrations of LiCl and harvested at 36-hr gastrula and 84-hr
pluteus stages for staining. Antibody staining was visualized with fluorescein-conjugated
goat anti-rabbit
secondary antibody and observed under a fluorescence microscope. As previously reported (Nocente-McGrath
et
c& 1989), control embryos stained intensely over the entire archenteron surface at the gastrula stage, with this
staining becoming less intense and restricted primarily
to the midgut by the pluteus larval stage (Figs. 2A and
2D). In all cultures exposed to LiCl, a delay in hatching
and gastrulation
was observed so that at 36 hr these
embryos had not initiated gastrulation
(Figs. 2B and
2C). The vegetal plate region of these embryos stained
intensely, and the domain of reacting cells appears
NOCENTE-MCGRATH,
MCISAAC,
AND ERNST
447
LX1 Alters Cell Fate in Sea Urchins
larger than that which was observed in untreated embryos at the same morphological
stage (about 28-30 hr,
Fig. 2G). In addition, there is an exaggerated flattening
of the vegetal plate in the embryos treated with 30 mM
LiCl (Fig. 2C). At 34 hr of development,
the embryos
treated with 15 mMLiC1 display intense staining of the
everted gut (Fig. 2E). Embryos cultured in 30 mMLiC1
show moderate staining of the two smaller spheres, confirming their endodermal origin (Fig. 2F). In the culture
treated with the highest concentration
of lithium
(60
mM), staining was observed in small groups of cells
within the larger masses present, but again, no obvious
organization was evident (data not shown).
To quantitate
the increase in endoderm tissue suggested by the photographs presented in Fig. 2, we dissociated control and lithium-treated
embryos into single
cell preparations
at the mesenchyme blastula stage,
counted the number of cells which react with antiEnd016 antibodies, and compared this number with the
total number of cells detected by counterstaining
with
DAPI. The data from these experiments
are summarized in Table 1. These results demonstrate that as the
concentration
of LiCl in the culture medium increases,
the proportion of cells expressing the endoderm-specific
marker also increases, nearly doubling at the highest
concentration
of LiCl tested. Furthermore,
the increased proportion of endoderm tissue is evident even
prior to the onset of gastrulation.
The Effect of LiCl on the Temporal
End016 mRNA
FIG. 1. Morphology of vegetalized embryos at pluteus larva stage.
Cultures were exposed to various concentrations of LiCl as detailed
under Materials and Methods. Embryos were fixed in 1% formaldehyde and photographed with the aid of a polarizing filter to highlight
the larval skeletal system. (A) Control embryos. The three-part gut
and skeletal system are well developed. Pigment cells can be seen
dispersed throughout the ectoderm. (B) The 15 mAf LiCl-treated embryos. Exogastrulated embryos have normal skeletons and pigment
cell distribution. (C) The 30 mM LiCl-treated embryos. Snowmanshaped embryos have abnormal skeletons and asymmetric pigment
cell distribution.
Exprwtion
of
The effect of LiCl on the temporal and quantitative
expression of End016 mRNA in the developing sea urchin embryo was characterized. Figure 3 shows the results of Northern blot analysis of End016 expression in
15- and 37-hr control and LiCl-treated
embryos. In both
treated and untreated embryos, End016 expression is
not detected at 15 hr, indicating
that the gene is not
activated prematurely
in vegetalized embryos. At 37 hr,
a stage of development when this gene is normally expressed at high levels, a concentration-dependent
increase in the level of End016 mRNA is observed in embryos treated with LiCl. The batch of eggs used in this
experiment required treatment with 60 mM LiCl to effect the completely vegetalized morphology pictured in
Fig. 1C.
In the present study, a generalized morphological
delay for embryos exposed to LiCl was observed. To examine the molecular characteristics
of this delay, the
Northern blot of control and LiCl-treated
RNA at 15 and
3’7 hr of development was rehybridized with a probe specific for the late class histone H2B gene (LH2B) (Maxson et al, 1983b). The late histone genes are normally
448
DEVEWPMENTAL
BIOLOGY
VOLUME
147, 1991
NOCENTE-MCGRATH, MCISAAC, AND ERNST
TABLE 1
FRACTION OF CELLS EXPRESSING ENDO~G PROTEIN
IN NORMAL AND VECETAIJZED EMBRYOS
@Cl]
Anti-End016 stained cells at
the m. hlastula stage
Relative
increase
OmM
15 mM
30 mM
60mM’
23%
26%
35%
40%
1.13
1.52
1.74
transcribed at very low levels during cleavage stage and
the mRNAs rise sharply in abundance during the period
from 14-16 hr postfertilization,
a time when the abundance of the early histone mRNAs declines rapidly.
While End016 levels are induced in LiCl-treated
cultures, LHZB expression is markedly lower (Fig. 3). Later
in development, equivalent levels of LHBB histone are
found in control and LiCl-treated
embryos (data not
shown). Comparison of End016 and LHBB signals reveals a differential response to LiCl in the expression of
a lineage-restricted
gene (Endol6) and a temporally regulated, nonlineage-restricted
gene (LHBB).
DISCUSSION
Both classical and modern studies have shown that
exposure to lithium during the early stages of development causes cells to adopt a more vegetal fate, and at the
molecular level, to express vegetal markers that they
would not otherwise synthesize. Since End016 was
found to be expressed exclusively in cells of a vegetal
lineage (endoderm), it was of interest to examine the
effects of LiCl exposure on End016 expression. Classical
studies on whole embryos treated with lithium reported
a graded response, and the data presented here for S.
pwpwatus
support these observations. Furthermore,
these data show that this response occurs at the molecular as well as the morphological
level. These results are
consistent with the work of Livingston and Wilt (1989)
detailing a LiCl dose-dependent response for the development of vegetal markers in isolated animal blastomere cultures. The indirect immunofluorescence
stain-
LiCl Alters Cell Fate in Sea Urchins
449
12345676
Endo 16
L H26
FIG. 3. The effect of LiCl on End016 transcript levels. RNA isolated
from control and LiCl-treated embryos was size fractionated on a 2.2
M formaldehyde-l%
agarose gel and blotted to nitrocellulose. The
End016 and late H2B messages were detected by hybridizing to the
filter q-labeled
cloned fragments of the respective genes. RNA isolated 15 hr postfertilization
was loaded in lanes l-4,37 hr postfertilization in lanes 5-8. The samples (5 pg each) were loaded in the following order: control (lanes 1, 5), 15 mM LiCl (lanes 2, 6), 30 mM LiCl
(lanes 3,7), 60 mMLiC1 (lanes 4,8). End016 mRNA expression was not
observed until posthatching (18 hr) and was present at much higher
levels after LiCl exposure. Late class histone gene expression was retarded in vegetalized embryos.
ing results provide molecular
confirmation
of the
identification
of endodermal tissue that relied on morphological traits in the older literature, clearly demonstrating a change in cell fate resulting from exposure to
LiCl. In addition, the whole mount and single cell staining experiments demonstrate that anti-End016 antibodies detect an increase in presumptive endoderm tissue
even prior to the onset of gastrulation.
This increase
could cause a mechanical disruption of gastrulation
that
might contribute to the exogastrula morphology exhibited by the lithium-treated
embryos.
The developmental
delay in lithium-vegetalized
embryos that we observed has previously been reported
and is probably directly related to a slowing of cleavage
rates (Hagstrom,
1963). Our results demonstrate
that
these effects are accompanied by a delay in histone gene
class “switching” that normally occurs during development (see Maxon et aZ., 1983a for review). This result is
somewhat surprising since the transition from early to
late class histone gene expression is known to be inde-
FIG. 2. Immunolocalization
of End016 in vegetalized embryos. Cultures were exposed to various concentrations of LiCl as detailed under
Materials and Methods. Embryos were collected at 28 hr (late mesenchyme blastula, panel G), 36 hr (midgastrula stage; A, B, and C) and at 84 hr
(pluteus larva stage; D, E, and F). Whole mounts were fixed and stained with anti-End016 antibodies as described under Materials and Methods.
(A, D) Control embryos. Entire archenteron appears to stain at the earlier stage (A). Intensity of staining decreases as development proceeds
(D). (B, E) The 15 mMLiCl-treated
embryos. Developmental program is delayed in treated embryos as evidenced by delayed gastrulation (B, C).
The vegetal plate region stains intensely, characteristic of the mesenchyme blastula stage seen in (G). Everted guts at the pluteus stage (E) also
stain intensely. (C, F) The 30 mMLiCl-treated
embryos. As in (B), gastrulation is also delayed in these embryos. Note the flattened shape of the
embryo in (C) and the larger sector of stained cells in comparison to control embryo at the same morphological stage (G). Midpiece and “head”
of the pluteus stage vegetalized embryo react with the anti-End016 antibodies, revealing the great expansion of endodermal tissue in these
embryos.
450
DEVELOPMENTAL
BIOLOGY
pendent of cell-cell contact (Arceci and Gross, 1980;
Brookbank, 1980), cell division (Brookbank, 1978; Harrison and Wilt, 1982), or cell lineage (Angerer et aL, 1985).
Clearly, lithium exerts multiple and complex effects on
developing embryos. The use of sensitive lineage-specific markers, such as Endol6, may help to further dissect this fascinating phenomenon.
We thank L. Angerer and R. Maxson for the histone LHZB probe
and John McGrath for critical reading of the manuscript. Supported
by a R.A.W. Career Advancement Award from the NSF and a Biomedical Research Support Grant to S.G.E.
REFERENCES
ANGERER, L., DE LEON, D., Cox, K., MAXSON, R., KEDES, L., KAUMEYER, J., WEINBERG, E., and ANGERER, R. (1985). Simultaneous
expression of early and late histone messenger RNAs in individual
cells during development of the sea urchin embryo. Deu. Biol. 112,
157-166.
ARCEICI, R. J., and GROSS, P. R. (1980). Histone gene expression: Progeny of isolated early blastomeres in culture make the same changes
as in the embryos. Science 209,607~609.
BERRIDGE, M. J., DOWNES, C. P., and HANLEY, M. R. (1989). Neural and
developmental actions of lithium: a unifying hypothesis. Cell 59,
411-419.
BROOKBANK, J. W. (1978). Histone synthesis by cleavage arrested sea
urchin eggs. Cell &Q&r. 7,153-158.
BROOKBANK, J. W. (1980). Effects of cordycepin and cell dissociation
on the synthesis of Hl histone by sea urchin embryos. CeU D$er. 9,
315-321.
CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., and RIJT~ER,
W. J. (1979). Isolation of biologically active RNA from sources
enriched in ribonucleases. Biochemistry 18,5294-5299.
DAVIDSON, E. (1989). Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: a proposed mechanism.
Development 105,421-445.
HAGSTROM, B. E. (1963). The effect of lithium and 0-iodobenzoic acid
on the early development of the sea urchin egg. Biol, BuU. 124,55-64.
VOLUME 147.1991
HARRISON, M. F., and WILT, F. H. (1982). The program of Hl histone
synthesis in S purpuratus embryos and control of its timing. J. Exp.
Zoo1 223,245-256.
HERBST, C. (1892). Experimentelle untersuchungen iiber den einfluss
der veranderten chemischen zusammensetzung des umgebenden mediums auf die entwicklung der tiere I teil. Versuche an Seeigeleiern.
Z. Wi.s.s.Zoo1 55,446-518.
HORSTADIUS, S. (1973). “Experimental Embryology of echinoderms.”
Clarendon Press, Oxford.
KAO, K. R., MASUI, Y., and ELINSON, R. P. (1986). Lithium induced
respecification of pattern in Xenopus la&s embryos. Nature 322,
371-373.
LALLIER, R. (1964). Biochemical aspects of animalization and vegetalization in the sea urchin embryo. Adv. Mwphogex 3,147-196.
LIVINGSTON, B. T., and WILT, F. H. (1989). Lithium evokes expression
of vegetal-specific molecules in the animal blastomeres of sea urchin embryos. hoc NatL Acad Sci. 86,3669-3673.
LIVINGSTON, B. T., and WILT, F. H. (1990). Range and stability of cell
fate determinations in isolated sea urchin blastomeres. Develop
ment 108,403-410.
MANIATIS, T., FRITSCH, E., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
MAXSON, R., MOHUN, T., COHN, R., and KEDES, L. (1983a). Expression
and organization of histone genes. Annu. Rev. Gen 17,239-277.
MAXSON, R., MOHUN, T., GORMENZANO, G., CHILDS, G., and KEDES, L.
(198313). Distinct organizations and patterns of expression of early
and late histone gene sets in the sea urchin. Nature 301,120-125.
NOCENTE-MCGRATH, C., BRENNER, C. A., and ERNST, S. G. (1989).
Endol6, a lineage specific protein of the sea urchin embryo, is first
expressed just prior to gastrulation. Dev. Bill 136,264-272.
PETERS, D. J. M., VAN LOOKEREN CAYPAGNE, M. M., VAN HAASTERT,
P. J. M., SPEK, W., and SCHAAP, P. (1989). Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum. J. Cell Sci. 93,205-210.
P~rraa~, D., and ERNST, S. G. (1984). Developmental time, cell lineage, and environment regulate the newly synthesized proteins in
sea urchin embryos. Dew. Biol. 106,236-242.