Download Lithium changes the ectodermal fate of individual frog blastomeres

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Development 106, 599-610 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Lithium changes the ectodermal fate of individual frog blastomeres because
it causes ectopic neural plate formation
STEVEN L. KLEIN and SALLY A. MOODY
Department of Anatomy and Cell Biology, University of Virginia Health Sciences Center, Box 439 Medical Center, Charlottesville, VA 22908,
USA
Summary
Amphibian blastulae that are treated with lithium (Li)
develop into embryos that consist almost exclusively of
head structures. This dramatic change in embryogenesis
may occur either because Li selectively kills trunk
progenitors or because Li causes trunk progenitors to
become head progenitors. To distinguish between these
possibilities, we compared the fates of individual frog
blastomeres between Li-treated embryos and normal
embryos using lineage tracers. The results demonstrate
that Li causes ventral midline cells, which normally
populate large amounts of trunk, to produce many head
structures, including the brain. Examination of fluorescently labeled clones in living Li-treated gastrulae
shows that: (1) the ectodermal members of the clones
migrate normally, and chordamesodermal involution
begins normally; (2) the chordamesoderm's later in-
volution is altered such that it is confined to the vegetal
hemisphere; (3) accordingly, the neural plate forms in
the vegetal hemisphere, circumscribing the blastopore,
which normally gives rise to the cloaca; and (4) the
ectodermal progeny of the ventral midline blastomeres
that are near the blastopore populate the brain because
they are induced by the stalled chordamesoderm to form
part of the ectopic neural plate. These results demonstrate that Li, administered during a short developmental window at early cleavage stages, ultimately alters
ectodermal fate because it changes the pattern of chordamesodermal involution during gastrulation, which in
turn changes the site of neural plate formation.
Introduction
olution of this important issue may indicate the mode of
action of Li-teratogenesis, the way in which cleavagestage manipulations alter cell fate, and the mechanisms
that control cell fate during normal development.
In this study, we marked single blastomeres of early
cleavage stage Xenopus laevis embryos with lineage
tracers, exposed the embryos to Li, and compared the
distribution of their progeny to that of normal embryos
(Moody, 1987a,b). We show that Li causes large
changes in cell fate, especially in ectodermal descendants, and that these changes result from alterations in
the pattern of gastrulation.
Lithium (Li), a known teratogen (Briggs et al. 1986),
suppresses trunk formation and enhances head formation when it is administered briefly to cleavage-stage
frog embryos (Kao et al. 1986; Breckenridge et al. 1987;
Condie & Harland, 1987; Regen & Steinhardt, 1987;
Kao & Elinson, 1988,1989; Cooke & Smith, 1988; Slack
etal. 1988; Klein & King, 1988; Busa & Gimlich, 1989).
The gross morphology of Li-treated embryos (Fig. 1A)
clearly illustrates that a major change in embryonic cell
fate has occurred. This change may result from alterations in cleavage stage intercellular communication
(Kao et al. 1986; Kao & Elinson, 1988), because Li is
effective only when administered during cleavage stages
(Kao et al. 1986; Breckenridge et al. 1987; Cooke &
Smith, 1988) and because it blocks the inositol trisphosphate-diacylglycerol second messenger pathway (Berridge et al. 1982; Sherman et al. 1981; Berridge, 1987;
Busa & Gimlich, 1989). One of the most striking fate
changes is a doubling of the number of neurons (Breckenridge etal. 1987). However, previous studies have not
indicated whether Li causes non-neuronal progenitor
cells to produce neurons, or whether the normal neuronal ancestors overproduce their normal progeny. Res-
Key words: cell fate, gastrulation, lithium treatment,
lineage tracers.
Materials and methods
Embryos of Xenopus laevis were obtained, dejellied, selected
and injected with lineage tracers as described by Moody
(1987a). One blastomere per embryo was labeled by microinjection of lnl of either horseradish peroxidase (HRP,
Boehringer Mannheim) or fluorescein-dextran-amine
(FDA, Molecular Probes). Embryos were incubated in
300mM-LiCl for a 6min interval during the 32-, to 128-cell
stage, followed by extensive rinsing in Steinberg's solution.
This treatment produced a majority (up to 90 %) of head-only
embryos with normal faces and heads (Fig. 1A), which met
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5. L. Klein and S. A. Moody
the criteria of DAI 7 and 8 (dorsoanterior enhancement
index) of Kao & Elinson (1988), in which DAI 5 are normal
and DAI 10 are radially symmetric dorsoanterior embryos.
When control embryos reached stage 33-43 (Fig. ID; Nieuwkoop & Faber, 1964), the Li-embryos were fixed, cryostat
sectioned and processed for either histochemistry (diaminobenzidine reaction for HRP; Moody, 1987a) or for fluorescence (FDA) by conventional methods.
Single identified blastomeres along the dorsal and ventral
midline of 16-, and 32-cell embryos were injected with the
lineage tracers. The positions of these blastomeres are illustrated in Figs 2 and 3. The dorsal midline blastomeres (Dl.l
and D2.1) of the normal 16-cell embryo are major head
progenitors. Both blastomeres contribute progeny to all head
mesoderm and endoderm, and Dl.l contributes heavily to all
head ectoderm, including the brain (Moody, 1987a). Of the
ventral midline blastomeres, V2.1 contributes virtually no
progeny to the head, and Vl.l's only head progeny populate
branchial arch mesoderm and non-central nervous system
ectoderm (Moody, 1987a). The distribution of the labeled
progeny within each organ of the Li-embryo was mapped
from complete serial sections, as described previously for
normal embryos (Moody, 1987a).
Fate maps identical to those constructed for normal embryos were made for Li-treated embryos. In order to minimize
population variations (see Moody, 1987ft), at least 10 embryos
per blastomere were analyzed. Although these DAI 7/8
embryos were 'dorsoanterior enhanced' (Kao & Elinson,
1988), a small amount of rostral trunk persisted. For our
analysis, we considered an organ to be a trunk structure if it
was located posterior to the otocyst in transversely sectioned
material.
Complete fate maps were constructed for 86 HRP-labeled
Li-embryos. The relative proportion of labeled progeny in
each organ was assessed in coded specimens as described
previously (Moody, 1987a). Quantitative estimates of cell fate
were made by assigning a numerical value to each region of
each organ as follows: no labeled cells = zero; five or fewer
labeled cells = 0-5; many labeled cells = 1-5; almost completely labeled = 3-0. The relative contribution of the blastomere to each organ was determined by summing the value of
each organ region and averaging the numerical value of the
organ over all specimens in which that blastomere had been
injected. Using this procedure, the average amount of central
nervous system (CNS) that is produced by a normal VI. 1 is:
forebrain, 0-17 (S.E. =0-17); midbrain, 006 (0-06); hindbrain, 0-05 (0-05); spinal cord, 1-3 (0-13). The average
amount of CNS produced by a Li-treated VI. 1 is: forebrain,
0-86 (0-64); midbrain, 1-84 (0-48); hindbrain, 3-63 (0-49);
anterior spinal cord, 3-47 (0-35). The posterior spinal cord
was not present in the DAI 7/8 embryos. The difference
between the fate of the Li-treated blastomere and the normal
blastomere was determined by subtracting the normal average
value from the Li-average value (Fig. 2). Differences of ±1
were considered to be significant because they distinguish
between a few and many labeled progeny.
To determine whether the CNS progeny of ventral blastomeres were actually neurons, many FDA-labeled embryos
were prepared for indirect immunofluorescent localization of
neurofilament proteins. Frozen tissue sections were incubated
in a monoclonal antibody directed against the phosphorylated
high and middle molecular weight atxon-specific neurofilament proteins (Sternberger-Meyer; SMI 31 at a 1:50000
dilution), and a goat anti-mouse secondary antibody conjugated to RITC (HyClone at a 1:100 dilution).
We examined whether Li treatment altered the pattern of
gastrulation by monitoring the position of FDA-labeled
clones in both living and fixed specimens. During gastrulation, neurulation and tail bud stages, the location of the
superficial members of the FDA-labeled clones of living
embryos (33 normal and 34 Li-treated embryos that developed to DAI 8) were examined continuously with a Wild M5
stereomicroscope equipped for epifluorescence and an image
intensifier (KS-1380; Videoscope International, Ltd). The
positions of both the superficial and deep members of the
clones also were examined by dissecting 43 normal and 42 Liembryos that were fixed between the beginning of gastrulation and the tail bud stages.
Finally, in order to confirm the ectopic position of the
neural plate, 35 uninjected normal and 43 uninjected Liembryos were fixed and examined between neurula and
tailbud stages. The blastopore lip of these gastrulae were
marked with a spot of the vital dye Nile blue sulfate
(Kirschner & Hara, 1980). The dye was applied to the dorsal
blastopore lip of the normal embryos, and to a portion of the
Li-blastopore lip. However, we could not identify the specific
region of the Li-blastopore lip that was labeled because the lip
is not visibly polarized in Li-embryos.
Results
Lithium-treated ventral blastomeres populate the
central nervous system
The changes in cell fate that the Li-treatment produced
were most obvious in the CNS. The two dorsal blastomeres, which contribute to CNS normally, continued to
do so. However, the two ventral blastomeres, which
normally make significant contributions to only the
dorsal spinal cord, contributed numerous progeny to
the CNS. The VI. 1 blastomere (ventral animal midline
cell) showed the most dramatic change in cell fate. In
the normal embryo, the ectodermal progeny of VI. 1
populate large amounts of epidermis in both the head
and the trunk; they populate virtually none of the brain
(Fig. IE), and only a small amount of the dorsal spinal
cord (Moody, 1987a). But, in the Li-treated embryo,
Vl.l's progeny populated large areas of brain and
retina (Fig. IB, C). Many of these progeny had a
neuronal morphology, with long axon-like processes,
most of which were in established axonal tracts such as
the ventral longitudinal tract of the forebrain and the
ventral midbrain commissure (Fig. 1C). The presence
of neurofilaments in these FDA-labeled fibers verified
that they were axons (Fig. 1C, inset). Thus, in Litreated embryos, the progeny of VI. 1 occupy an
abnormal location (head rather than trunk) and have an
abnormal phenotype (neuron rather than epidermis).
To estimate the extent of the fate changes in the
ectodermal derivatives, and to determine whether less
obvious changes in fate occurred in other organ systems, the relative contribution that each blastomere
made to each organ system was given a numerical value,
which was compared to values similarly derived from
the normal fates of the same blastomeres. Fig. 2 illustrates several significant fate changes in each of the
midline blastomeres. VI. 1 populated increased
amounts of head and trunk epidermis, otocyst, cranial
ganglia, midbrain, hindbrain and anterior spinal cord.
Additionally, VI. 1 produced smaller amounts of hind-
Lithium changes neural plate location
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Fig. 1. (A) Examples of Li-treated embryos in which the trunk failed to develop (DAI 8 of Kao & Elinson, 1988). Each has
a well-developed face and head including cement gland (c), stomqdeum (s), olfactory pits (o) and eyes (e). (B) A section
through the level of the eye of a Li-treated embryo in which the VI.1 blastomere was injected with HRP. Many of the VI.1
progeny populate the forebrain (b) and retina (r). n, neurocoele; nt, notochord. (C) A fluorescence photomicrograph of a
section through the midbrain of a Li-treated embryo in which VI. 1 was labeled with FDA. Many FDA-labeled axons are in
the ventral commissure (arrowheads). The inset shows that these same fibers also are immunoreactive for neurofilament
proteins. The arrowheads are in the same positions as those in the FDA micrograph, n, neurocoele; g, fifth cranial ganglion.
(D) A control embryo of the same age as those in A (stage 41 of Nieuwkoop & Faber, 1964). (E) A section through the
level of the eye of a control embryo in which VI.1 was labeled with HRP. No labeled progeny are present at this axial level.
The dark spots are melanin granules, not HRP. r = retina; b, forebrain; n, neurocoele.
gut. V2.1 (ventral vegetal midline cell) produced
increased amounts of midbrain, hindbrain, head
somite, foregut and anterior spinal cord, and reduced
amounts of trunk epidermis, trunk neural crest, trunk
somites, nephrotome and hindgut. Dl.l (dorsal animal
midline cell) produced increased amounts of head
epidermis, forebrain and midbrain, and reduced
amounts of foregut and hindgut. D2.1 (dorsal vegetal
midline cell), which normally produces very small
amounts of ectoderm (Moody 1987a), did not alter its
ectodermal fate (except for a reduced contribution to
the greatly stunted Li-spinal cord). However, D2.1
produced smaller amounts of mesodermal and endodermal structures in both head and trunk regions.
Dl.l normally contributes many progeny to the CNS,
and after Li-treatment this contribution was increased
significantly (Fig. 2). In contrast, VI. 1 and V2.1 normally contribute very few progeny to the CNS. The
large increase in their CNS contribution after Li-
treatment (Fig. 2) may have resulted either from extra
cell divisions of the few ventral progeny that normally
populate the CNS, or from an actual change in the fate
of descendants that normally produce other phenotypes. To distinguish between these possibilities, we
mapped the fate of the daughters of these two ventral
midline blastomeres. In normal embryos, the equatorial
daughters of VI.1 and V2.1 (VI.1.2 and V2.1.2,
respectively) both contribute to CNS, whereas the polar
daughters (VI. 1.1 and V2.1.1, respectively) have no
CNS descendants (Moody, 19876). Specifically, VI. 1.1
has no descendants in the forebrain or midbrain and
contributes an average amount of only 0-045 (i.e. a total
of less than 5 cells in eleven embryos) to the hindbrain,
and V2.1.1 has no descendants in any brain region (c.f.
Dale & Slack, 1987). Following Li-treatment, the equatorial daughters increased their CNS progeny more
than the polar daughters (Fig. 3). In fact, V2.1.2 was
responsible for the entire change seen in V2.1's fate
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Fig. 2. Histograms showing the extent to which each structure of the Li-treated embryo contains progeny of the indicated
blastomere. The diagram in the left corner of each graph shows the location of the injected blastomere (blackened) in a 16cell embryo with the animal pole up and dorsal midline to theright.The graphs show the contribution of the Li-blastomere
minus the contribution of the normal blastomere (normal data from Moody, 1987a). Positive values indicate a greater than
normal contribution by the Li-blastomere and negative values indicate a smaller than normal contribution by the Liblastomere. The dotted lines at +1 and at - 1 indicate significant differences from normal. The structures are arranged in
roughly anterior (left) to posterior (right) order; the group of structures on the left of each graph is in the head and that on
the right is in the trunk. The interface between the head and the trunk was considered to be the level of the otocyst.
According to this criterion, DAI 7/8 embryos contain a small amount of anterior trunk structures. Gl, gland; Epi,
epidermis; Olfactory, olfactory pit; Cr, cranial; D head Mes, dorsal head mesoderm; Br, branchial; Hd, head; Tr, trunk; Cd,
cord; Lat Plate, lateral plate mesoderm.
map, indicating that Li-treatment caused the progeny of
V2.1 that normally populate the CNS to produce extra
CNS. However, the polar daughter of VI. 1 (i.e.
VI. 1.1) also made a large contribution to all parts of the
CNS. This is a significant change in cell fate because
VI. 1.1 normally produces only a few of the spinal cord's
Rohon-Beard neurons (Moody, 19876; Moody, 1989),
which are derived from the neural crest and not from
the neural plate (Du Shane, 1938; Chibon, 1966). Thus,
the fate changes of VI. 1.1 show that Li-treatment
causes a non-CNS progenitor to produce CNS.
Changes in ectodermal fates are correlated with
changes in the pattern of gastrulation
Because Li-treated ventral midline blastomeres
changed their fate to become major neuronal progenitors, it seemed likely that Li perturbs a cell-cell
communication that either directly influences cell fate
(as suggested by Kao et al. 1986; Kao & Elinson, 1988;
Slack et al. 1988; Busa & Gimlich, 1989), or that
indirectly influences cell fate by altering the pattern of
gastrulation such that novel cell populations, i.e. the
progeny of ventral blastomeres, come under the influence of Spemann's Organizer (i.e. the presumptive
chordamesoderm; Spemann & Mangold, 1924; Spemann, 1938). To distinguish between these possibilities,
we studied the gastrulation movements of fluorescently
labeled clones of Dl.l and VI. 1 in living Li-embryos
that developed to DAI 8. In all cases, the migration of
the superficial normal and Li-clones of each blastomere
was indistinguishable. For example, the progeny of
both normal and Li-Vl.l migrated ventrally so that at
the end of gastrulation they each occupied the same
mostly unilateral strip between the ventral tip of the
animal pole and the ventral Up of the blastopore
(Fig. 4A, B, F and summarized in Fig. 5).
Lithium changes neural plate location
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Structures
Fig. 3. Differences from normal contributions of the daughters of VI. 1 (A) and V2.1 (B) to the Li-CNS (normal data from
Moody, 19876). V2.1's equatorial daughter cell, V2.1.2, accounts for almost all of the change of fate seen for the mother cell
(left panel in B), and Vl.l's equatorial daughter cell, VI.1.2, accounts for more of the change in cell fate seen for the
mother cell (left panel in A). However, Vl.l's anterior daughter (VI.1.1) changes fate to produce greatly increased amounts
of CNS. See the legend of Fig. 2 for abbreviations.
Nevertheless, the processes of gastrulation and
neurulation were retarded and abnormal in the Liembryo. Marking the blastopore lip with a spot of Nile
blue sulfate toward the end of gastrulation (stages
11-5-12) labeled the posterior third to half of normal
embryos (c.f. Keller, 1975), but labeled the rostral tip of
the DAI 8 Li-head (data not shown). This result
suggested that the Li-head formed around the blastopore, which normally becomes the cloaca (Fig. 5). This
proposal was confirmed by observing neural plate
formation in Li-embryos. First, a dark line formed
around the yolk plug and extended to the equator,
where the cement gland formed (Fig. 4C). The ectoderm within this line formed an ovoid plateau
(Fig. 4G). The part of the plateau away from the
cement gland contained a large number of VI. 1
progeny, and the side of the plateau near the cement
gland contained a small VI.1 subclone (Fig. 4E, G).
The part of the plateau near the cement gland contained
many D l . l progeny. The edges of the plateau eventually raised up and fused to form a short tube with the
cement gland at one end. The head subsequently
formed from this region of the Li-embryo. The ectodermal plateau that formed around the blastopore, and
contained many VI. 1 progeny (Fig. 4E, G), was obviously the Li-neural plate (c.f. Kao & Elinson, 1988).
Fig. 5 summarizes the position of the fluorescent Dl.l
and VI. 1 clones that we observed in living embryos at
different stages of development.
Examination of the interior of Li-embryos during
gastrulation and neurulation demonstrated that the
neural plate formed around the blastopore, instead of
along the dorsal midline, because the chordamesoderm
involuted abnormally. Although the involuting chordamesoderm began to migrate beneath the dorsal ectoderm as in the normal embryo, its leading edge stopped
when it reached the equator (Figs4D, 6B, C); normally, the leading edge migrates to the animal pole
(Fig. 6A; and Nieuwkoop & Faber, 1964). Then, the
involuting ventral mesoderm, instead of migrating beneath the ventral ectoderm (Keller, 1975), joined the
posterior chordamesoderm deep to the yolk plug
(Fig. 7). The combined (i.e. dorsal and ventral) mesodermal finger moved into the center of the embryo to
become the 'internal proboscis' described by Kao &
Elinson (1988) (Figs 4D, E, 6B, C). The chordamesoderm folded onto itself; its anterior portion was directly
beneath the dorsal vegetal ectoderm and its posterior
portion was beneath the periblastoporal ectoderm and
in the embryo's interior (Figs 4D, 6B, C). The anterior
ventral mesoderm was in the proboscis and the posterior ventral mesoderm was beneath the periblasto-
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S. L. Klein and S. A. Moody
Fig. 4. (A, B) The vegetal hemisphere of the gastrula showing the location of the FDA-labeled VI.1 clone. Ventral is to the
left. (A) Li-embryo, (B) normal embryo (stage 11-5), y, yolk plug. (C) The vegetal hemisphere of a Li-neurula showing the
neural plate, delineated by the dark line, around the yolk plug (arrow). The cement gland (c) is at the rostral end of the
line, and indicates the dorsum of the embryo. (D) The interior of the same embryo shown in C following a midsagittal
dissection. The yolk plug (arrow in C) was lifted from the surface of the neural plate (between the arrowheads). The animal
pole (an) is to the bottom of the figure and the cement gland, i.e. the dorsal side of the embryo, is to the right. The
involuted chordamesoderm occupies two areas within the original vegetal hemisphere. Its leading edge (+) extends beneath
the dorsal ectoderm about 90° from the yolk plug, and the remainder forms a portion of the 'internal proboscis' (bracket).
The neural plate forms superficial to both areas of chordamesoderm and invaginates beneath the yolk plug to form the core
of the proboscis. (E) Fluorescence photomicrograph of the same embryo shown in D. FDA-labeled VI.1 progeny are
located in the ventral epidermis (e), in the ventral half of the internal proboscis (p), in the neural core of the proboscis
(arrow) and in the neural plate (arrowheads). (F) A side view of a Li-neurula showing the location of the FDA-labeled VI. 1
clone. A large external yolk plug is at the top of the figure (between the arrowheads) and the cement gland (c) is to the right
of the yolk plug. (G) A top view of the same embryo shown in F following the removal of the yolk plug. Beneath the yolk
plug is the neural plate (border outlined by arrowheads) containing many FDA-labeled VI. 1 progeny. A small portion of the
clone (arrow) is in the anterior part of the neural plate.
poral ectoderm. The neural plate formed from the
dorsal and ventral ectoderm that was superficial to the
stalled, vegetal, chordamesoderm (Figs 4D, E, 6B, C).
The posterior ventral mesoderm and some of the
posterior chordamesoderm were located in the presumptive head. This altered pattern of gastrulation is
summarized in Fig. 7.
The neural plate formed from the ectoderm that was
superficial to the chordamesoderm. The leading edge of
the chordamesoderm denned the anterior extent of the
neural plate in all embryos regardless of the extent of
dorsoanterior enhancement. Neurulae in which the
neural plate was of normal or near-normal length
developed to DAI 5 or 6 (normal or stunted tail). In
most of these neurulae, the chordamesoderm had
migrated beneath the dorsal ectoderm from the dorsal
lip of the blastopore to the anterior pole. In those
neurulae in which chordamesodermal involution was
incomplete, a small knob of mesoderm projected into
the archenteron from the caudal pole. Conversely,
neurulae with a round neural plate circumscribing the
blastopore developed to DAI 9 or 10 (radially symmetric dorsoanterior). In these neurulae, the chordamesoderm had migrated only a small distance from the
blastopore beneath dorsal ectoderm; the remainder of
the chordamesoderm formed a large internal proboscis.
Thus, the extent of dorsoanterior enhancement was
inversely related to the amount of chordamesoderm
beneath ectoderm, and was directly related to the size
of the internal proboscis.
Discussion
The determination of embryonic cells probably occurs
in several steps, and can be influenced by several
extracellular cues. Among the interactions considered
Lithium changes neural plate location
605
influence gastrulation, and that the altered pattern of
gastrulation changes cell fate secondarily.
Fig. 5. Diagram summarizing the gastrulation movements
of the superficial members of the VI.1 (dots) and Dl.l
(stippling) clones in normal and Li-treated embryos. In
each embryo, the animal pole is to the left. The ectodermal
progeny migrate normally during gastrulation so that they
occupy a strip between the animal pole (left) and the lip of
the blastopore (right), y, yolk plug. The members of each
of the Li-clones that are near the blastopore populate the
CNS because the neural plate (np, outlined by the thin
internal line in the neurula) forms from the periblastoporal
ectoderm. Some of the periblastoporal members of the
VI. 1 clone migrate onto the dorsum of the neurula
(dorsally located dots on the caudal part of the normal
neurula and in the anterior neural plate of the Li-neurula).
The Li-larva forms upside down; the vegetal members of
the VI.1 and Dl.l clones that normally populate the caudal
embryo populate the Li-head, and the animal members of
the VI.1 and Dl.l clones that normally populate the head
populate the caudal regions of the Li-embryo. The VI. 1
progeny that produce the dorsal epidermis of the normal
larva are 'removed' to show the contributions of Dl.l to
the deeper dorsal structures, i.e. brain and spinal cord. D,
dorsal; V, ventral.
to be influential are intercellular communications via
growth factors, morphogens and/or inductors (Slack,
1983). It has been well documented that administration
of Li during cleavage stages produces dramatic changes
in embryogenesis (Kao et al. 1986; Breckenridge et al.
1987; Condie & Harland, 1987; Regen & Steinhardt,
1987; Kao & Elinson, 1988,1989; Cooke & Smith, 1988;
Slack et al. 1988; Klein & King, 1988; Busa & Gimlich,
1989), and several laboratories have concluded that
these changes indicate that cell fate is influenced
directly by an early Li-sensitive event (Kao et al. 1986;
Kao & Elinson, 1988, 1989; Slack et al. 1988; Busa &
Gimlich, 1989).
To test this proposal directly, we used lineage tracers
to precisely compare the fates of individual identified
blastomeres between normal and Li-treated embryos.
We demonstrate that incubation in Li causes significant
changes in cell fate, especially in ventral, midline
blastomeres. The most dramatic changes were the gross
overproduction, and novel expression, of CNS
progeny. These changes in cell fate can be attributed to
alterations in the pattern of gastrulation, which brought
novel cell populations into contact during primary
embryonic induction. Therefore, we propose that Li
primarily perturbs cleavage-stage events that ultimately
Changes in cell fate
The DAI 7/8 embryos that were examined in the
present study provide a very useful tool for cell fate
studies because they primarily consist of only a properly
organized head. The absence of body structures would
result if the Li-treatment selectively killed the trunk
progenitors or if the treatment caused trunk progenitors
to produce head structures. Fate maps of 16-cell embryos can help to distinguish between these possibilities
because the potential to make head versus trunk is
largely segregated by this stage. Head structures are
populated largely by dorsal blastomeres and trunk
structures are populated largely by ventral blastomeres
(Moody, 1987a). The fate maps show that Li causes the
ventral blastomeres to produce significantly decreased
amounts of trunk and significantly increased amounts of
head structures. Additionally, continuous observation
of fluorescent clones during gastrulation and neurulation detected no obvious cell death in over 95 % of the
embryos. Therefore, Li-treatment appears to effect
specific changes in cell fate, rather than cause the
selective death of trunk progenitors.
Because each frog blastomere gives rise to a very
large number of progeny, and because their progeny
reside in a several organs and spatial locations, their
fates are difficult to quantify. Only a few cell populations consist of sufficiently small numbers to enable
cell counting (e.g. Moody, 1989). Therefore, we could
only estimate the changes in clone size in each organ by
constructing semiquantitative fate maps of Li-embryos
in a manner identical to that used for normal embryos
(Moody, 1987a,fr). Comparison of the Li- and the
normal semiquantitative fate maps show relative differences from normal cell fate. Because these differences
are only estimates, they should be interpreted conservatively, and we consider only large changes (>1) to be
significantly different from normal. In spite of these
potential difficulties in quantification, many striking
changes in cell fate were observed after Li-treatment.
The 16-cell fate maps show that Li-treatment causes
an animal hemisphere cell (Dl.l), which normally
contributes large numbers of progeny to the CNS, to
increase significantly its contribution to forebrain. Litreatment also causes two ventral blastomeres, which
normally contribute ectodermal progeny only to epidermis and to small areas of the dorsal spinal cord, to
significantly increase their contribution to brain and
spinal cord. In fact, the anterior blastomere (VI. 1)
becomes a major progenitor of neurons in the brain.
These increases in CNS descendants may have resulted
either from extra cell divisions of the normal neuronal
progenitors (as in nematode reiterative lineage mutants; reviewed in Sternberg & Horvitz, 1984), or from
the recruitment of extra CNS progenitors from blastomeres that do not normally populate the CNS (as in
Drosophila neurogenic mutants; reviewed in CamposOrtega, 1988). We could not distinguish between these
possibilities directly with the 16-cell fate maps, because
606
5. L. Klein and S. A. Moody
Fig. 6. Midsagittal view of normal (A) and Li- (B and C)
embryos at the end of gastrulation. Both normal and Liembryos are oriented the same way; the vegetal pole is to
the left (y, yolk plug). (A) The leading edge of the normal
chordamesoderm (+) migrates about 180°, to the animal
pole (*), and the ectoderm between it and the yolk plug
becomes the neural plate (dark ectoderm between
arrowheads). The anterior thickening of the neural plate
will form the brain. The anterior somites are segmented
already (bracket). (B) The leading edge of the Lichordamesoderm (+) migrates about 90°, to the equator.
The remainder of the Li-chordamesoderm moves into the
center of the embryo (0, its leading edge). The neural plate
is between the white arrowheads. The anterior part of the
Li-neural plate forms from the ectoderm that is between the
chordamesoderm's leading edge and the yolk plug (y),
whereas the posterior part of the neural plate forms from
the ectoderm on the ventral side of the yolk plug. The
rostral somites are visible in the vegetal hemisphere
(bracket). (C) An example of an older Li-embryo with a
more extensive internal proboscis. The first few somites of
the Li-embryo (bracket) form from the mesoderm in the
vegetal dorsal quadrant.
each of these blastomeres normally has a small, but
demonstrable, contribution to the CNS. However, fate
maps of the 32-cell stage can be used to make this
distinction because at this stage some blastomeres
normally have no CNS progeny (Jacobson & Hirose,
1981; Gimlich & Cooke, 1983; Jacobson, 1984; Dale &
Slack, 1987; Moody, 19876,1989). Analyses of the CNS
contribution by Li-treated 32-cell-stage ventral midline
blastomeres demonstrate that the largest increase in
CNS progeny descend from the equatorial daughters,
i.e. from blastomeres that normally contribute at least a
few cells to CNS. Thus, Li appears to cause those
blastomeres that produce neurons normally to produce
them in greater numbers, possibly by extra cell divisions. Additionally, Li causes at least one non-CNS
progenitor to contribute progeny to the CNS. Normally, Vl.l.l's only ectodermal progeny are epidermis
and neural crest (including the Rohon-Beard neurons,
which are embedded in the dorsal roof of the neural
tube, Moody, 19876); after Li-treatment, this blastomere produces large numbers of neurons throughout
the brain and spinal cord.
These fate changes represent a shift from the ventral
to the dorsal ectodermal phenotype. They were demonstrable because the ectodermal derivatives display a
very different phenotype (epidermis vs CNS) depending on their location in the tail bud embryo (ventral vs
dorsal). Comparable changes in mesodermal and endodermal fates were not detected. However, because
most mesoderm and endoderm display similar phenotypes in both ventral and dorsal locations, such changes
may not be detectable by fate mapping. Thus, our fate
maps do not enable one to conclude whether mesoderm
and endoderm are shifted from ventral to dorsal locations. However, our analyses did demonstrate
changes in the posterior-anterior locations of mesodermal and endodermal descendants. For example, at least
one blastomere (V2.1) that normally contributes to
only trunk mesoderm and endoderm (Moody, 1987a;
Dale & Slack, 1987), populates these tissues in the head
of the Li-treated embryo. Additionally, the Li-head was
populated by blastomeres (Dl.l and VI. 1) that normally contribute to these tissues in both the head and
the trunk. This change in posterior-anterior position of
mesoderm and endoderm may represent a specific
alteration in cell fate (Cooke & Smith, 1988), but more
probably results from the reorganization of the
anterior-posterior axis of the Li-embryo that occurs
because the reduction in the extent of gastrulation
Lithium changes neural plate location
Fig. 7. Diagram illustrating the formation of the internal
proboscis. The midsagittal plane of the embryo is depicted
and oriented as in Fig. 6. Asterisk indicates the animal
pole. The mesoderm is blackened; the chordamesoderm is
originally on the dorsal side (top in A) and the ventral
mesoderm is originally on the ventral side (bottom in A).
(A,B) Li-gastrulation begins normally. The leading edge of
the chordamesoderm (+) migrates beneath the dorsal
ectoderm. (C) The leading edge of the chordamesoderm
stops when it reaches the equator and the rest of the
chordamesoderm (O) moves into the interior of the
embryo. (D) The posterior chordamesoderm unites with the
ventral mesoderm beneath the yolk plug (y), and the
combined mesodermal finger moves into the center of the
embryo. (E) At the end of gastrulation, the
chordamesoderm is folded onto itself. The anterior portion
of the chordamesoderm is directly beneath the dorsal
vegetal ectoderm and the posterior portion is beneath the
periblastoporal ectoderm and in the embryo's interior. The
anterior portion of the ventral mesoderm is in the proboscis
and the posterior portion is beneath the periblastoporal
ectoderm. (F) The neural plate (np) forms from the
ectoderm that is superficial to mesoderm. c, cement gland.
causes the head to form in the vegetal hemisphere.
Because some of the 'posterior' mesoderm and endoderm also are located in the vegetal hemisphere at the
end of gastrulation, it differentiates into head, rather
than trunk, structures (i.e. V2.1 produces less hindgut,
nephric tubules and trunk somite, and produces more
foregut and head somite; see Fig. 2). Thus, our data
suggest that this change in endodermal and mesodermal
607
fate is the secondary result of alterations in the pattern
of gastrulation.
In addition to changes in the axial position of
mesodermal and endodermal descendants, there is
evidence that some mesodermal and endodermal
progeny are lost from the Li-treated embryo. Most of
the loss seems to result from the failure of the cells to
develop rather than to cell death. As mentioned above,
extensive cell death was not detected. In less than 5 %
of the embryos, the yolk plug remains external after
gastrulation (e.g. Fig. 4F), and falls off at hatching; in
only this small proportion of embryos does cell death
obviously contribute to the reduction in trunk structures. Conversely, nearly all blastomeres show significant decreases in hindgut progeny, and D2.1 shows
large decreases in mesodermal and endodermal
progeny without any corresponding increases in other
organs. Possibly some of the D2.1 stem cells die, or they
do not complete the normal number of cell divisions.
From these changes in cell fate, it appears that the trunk
of the Li-embryo fails to develop due to a combination
of the following: (1) the trunk mesodermal and endodermal progeny of V2.1 populate head, rather than
trunk, structures, (2) the hindgut progeny of VI.1 and
Dl.l fail to differentiate, and (3) some of the mesodermal and endodermal progeny of D2.1 either die or
divide less than normal.
It has been proposed that the Li-phenotype results
because Li causes ventral cells to behave as Spemann's
Organizer (Kao & Elinson, 1988). However, our fate
maps show that none of the midline blastomeres produces detectably increased amounts of the Organizer's
differentiated tissues (notochord or somite). Although
V2.1 produces more head somite, it produces correspondingly less trunk somite, suggesting a change in
axial position rather than a change in phenotype. In
contrast, fate maps of three cases of radially symmetric
dorsoanterior embryos (DAI 9/10) demonstrate that
V2.1 produces significantly more notochord than normal (data not shown), supporting the conclusion of Kao
& Elinson (1988) that the ventral marginal zone may
behave as an additional source of Spemann's Organizer
in DAI 9/10 Li-embryos.
Effects on gastrulation movements
The dramatic changes in cell fate that were caused by
Li-treatment during cleavage stages may indicate that
Li alters directly a cleavage-stage event that normally
influences cell fate. That is, Li may cause cells to
'transfate' (e.g. as in leech embryos, Weisblat & Blair,
1984). Alternatively, Li may change the spatial relationship between the ectoderm and the neural inductive
chordamesoderm during gastrulation. The latter
seemed the more likely possibility, especially because
ventrally transplanted presumptive chordamesoderm
causes ventral midline blastomeres to produce CNS
(Gimlich & Cooke, 1983; Jacobson, 1984).
Continuous observation during gastrulation showed
that the superficial ectodermal cells of the Li-treated
VI. 1 clone migrate normally. All of the cells of the VI. 1
clone that move migrate toward the ventral lip of the
608
S. L. Klein and S. A. Moody
blastopore; none of them migrate onto the dorsal side
of the embryo, into possible contact with a normally
located inductor. However, direct observation and vital
dye marking showed that the neural plate of the Liembryo forms ectopically. VI. 1 progeny are in the
neural plate, not because they migrate improperly, but
because the neural plate forms in the vegetal hemisphere, in part, from the ventral ectoderm that normally contains VI. 1 progeny. Previous studies also have
shown that the neural tissue of Li-treated embryos
forms near the blastopore (Kao & Elinson, 1988; Cooke
& Smith, 1988).
The ectopic neural plate is coincident with ectopic
chordamesoderm, which also is confined to the vegetal
hemisphere. Although chordamesodermal involution
begins normally, the leading edge of the chordamesoderm stops at the equator. The remainder of the
chordamesoderm and the adjacent ventral mesoderm
subsequently move into the interior of the embryo.
Thus, at the end of gastrulation, the anterior portion of
the chordamesoderm is deep to the dorsal vegetal
ectoderm, and the posterior portion is beneath the
periblastoporal (i.e. dorsal and ventral) ectoderm and
in the internal proboscis (summarized in Fig. 7). The
neural plate forms from the vegetal ectoderm that is
superficial to both regions of chordamesoderm; some of
this ectoderm derives from dorsal blastomeres and
some from ventral blastomeres.
The ventral periblastoporal ectoderm, which includes
VI. 1 and V2.1 progeny, is induced to form the posterior
neural plate (Figs 4C-G and 5), which probably differentiates into the spinal cord, hindbrain and midbrain.
The dorsal periblastoporal ectoderm, which includes
D l . l progeny, is induced to form the anterior neural
plate (Fig. 5), which probably differentiates into the
rest of the midbrain and the forebrain. This novel
location of the neural plate accounts for the observation
that VI. 1 populates more of the caudal CNS than
normal, whereas Dl.l populates more of the rostral
CNS than normal (see Fig. 2). The periblastoporal
location of the neural plate also explains why VI. 1 and
V2.1 produce increased amounts of spinal cord,
whereas D2.1 produce decreased amounts.
A normal secondary migration probably relocates
some of the VI. 1 progeny into the forebrain and retina.
Members of the VI. 1 clone migrate from the posterior
neural plate, around the blastopore and into the anterior neural plate on the dorsal side of the embryo
(Figs 4G, 5). In normal embryos, some ventral cells
migrate along this route at the end of gastrulation to
populate dorsal epidermis (Fig. 5; Keller, 1975). The
normal occurrence of this secondary migration further
suggests that the Li-treatment alters the involution of
the mesoderm without changing the movement of
ectodermal cells.
The reason that the posterior chordamesoderm
moves into the interior of the embryo, rather than
involuting beneath the dorsal ectoderm, may indicate
the primary action of Li-treatment upon gastrulation
movements. A lot of recent evidence shows that the
migration of the ventral mesoderm of Li-treated em-
bryos resembles that normally seen in dorsal mesoderm. The ventral mesoderm of Li-treated embryos
undergoes precocious involution so that the normal
dorsal-ventral asymmetry of involution is reduced
(Regen & Steinhardt, 1988). Additionally, ventral
mesoderm explanted from Li-treated embryo becomes
elongated as the cells undergo the movements of
convergent extension normally seen only in dorsal
mesoderm (Regen & Steinhardt, 1988; Slack etal. 1988;
Kao & Elinson, 1989). Considering that both dorsal and
ventral mesoderm of Li-treated embryos elongate, it
seems paradoxical that these embryos are stunted. The
reduction in overall length reflects the fact that the
chordamesoderm is folded onto itself. Therefore, the
folding of the chordamesoderm may be largely responsible for producing the morphology of the Li-treated
embryo. It may be that the Li-treatment also prevents
the leading edge of the chordamesoderm from entering
the animal hemisphere so that the posterior chordamesoderm is passively pushed into the interior by the
normal convergent extension of the dorsal cells (Keller
et al. 1985). Alternatively, the abnormal migratory
properties of the ventral mesoderm may cause it to
adhere to the posterior chordamesoderm and pull the
chordamesoderm into the interior of the embryo. The
crucial role of ventral mesoderm is also indicated by the
observation that injections of low doses of Li into
ventral blastomeres cause dorsoanterior enhancement
(Kao etal. 1986; Kao & Elinson, 1989; Busa & Gimlich,
1989).
•
In addition to acquiring the migratory properties of
dorsal mesoderm, the ventral mesoderm of Li-treated
embryos apparently acquires the ability to act as the
Organizer, because transplanted ventral marginal zones
from Li-treated gastrulae induce axis formation in u.v.irradiated axis-deficient embryos (Kao & Elinson,
1988). However, our fate maps show that ventral
midline blastomeres of DAI 8 embryos do not produce
increased amounts of the Organizer's differentiated
tissues (see above). It may be that some of the chordamesoderm in the internal proboscis is sufficiently close
to the ventral ectoderm to induce it to enter the CNS,
either by itself or in combination with the ventral
mesoderm. Regardless of whether the inductive signal
is derived from the dorsal and/or ventral mesoderm,
the reason that the CNS derives from only vegetal
ectoderm is that it is the only ectoderm that comes into
contact with the mesoderm.
These observations suggest that the Li-fate map
differs from normal because Li alters the involution of
the chordamesoderm and the ventral mesoderm. The
ectodermal fates are shifted toward head formation
because the vegetally stalled mesoderm induces the
formation of the nervous system from the vegetal
portion of the ectoderm on both the dorsal and ventral
sides of the blastopore. Accordingly, the dorsal and
ventral ectoderm of the vegetal hemisphere produce the
CNS, whereas the dorsal and ventral animal ectoderm,
which does not come into contact with the chordamesoderm (Fig. 4D), produces the epidermis of the remainder of the body (e.g. Li-Vl.l produced much more
Lithium changes neural plate location 609
trunk epidermis than normal, whereas Li-V2.1 produced much less trunk epidermis than normal, Figs 2
and 5). These changes represent both phenotypic and
spatial alterations in the ectodermal fates.
The observation that ventral cells are competent to
populate CNS is not novel. Previous studies have
demonstrated that transplantations that bring ventral
ectoderm into contact with presumptive chordamesoderm cause this ectoderm to produce nervous system
(Spemann & Mangold, 1924; Spemann, 1938; Gimlich
& Cooke, 1983; Jacobson, 1984). However, in the
present study, the manipulation that brought the ventral ectoderm into contact with the inductive mesoderm
was performed hours before gastrulation, and consisted
only of brief exposure to Li. This result shows that the
pattern of gastrulation is influenced by a Li-sensitive
event that occurs soon after fertilization. This event
may be involved in mesoderm induction, as suggested
by Kao & Elinson (1988, 1989) and by Slack et al.
(1988), and may then affect neural induction secondarily.
Action of lithium
Many biochemical studies have shown that Li blocks
the inositol trisphosphate-diacylglycerol second messenger pathway by inhibiting the conversion of inositol
phosphate to inositol (Sherman et al. 1981; Berridge et
al. 1982; Berridge, 1987). This pathway transduces
extracellular signals to intracellular calciumfluxesand,
thus, to changes in cell physiology. The observation that
Li alters pattern formation suggests that cleavage-stage
intercellular communications that are mediated by the
inositol trisphosphate pathway normally play a role in
later morphogenesis. In fact, recent studies have shown
that the detrimental effects of Li on embryogenesis are
reversed by injecting inositol into Li-treated embryos
(Busa & Gimlich, 1989). Additionally, Li reduces the
production of particular proteins that are synthesized
during cleavage stages (Klein & King, 1988). Thus, a Lisensitive cleavage-stage event, which may involve intercellular communication, may normally modulate the
physiological, and thus developmental, state of individual blastomeres. The present study shows that this
event influences the pattern of mesodermal involution
and thus ultimately influences the induction of the
nervous system. On-going examinations are expected to
reveal the nature of these cleavage-stage interactions
and the mechanism by which they influence gastrulation
movements.
We acknowledge the excellent technical assistance of
Daniel Best and Kathryn Kersey. Supported by NIH grants
HD23324 (SLK), and NS 23158 (SAM).
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