Download Fate maps of the first quartet micromeres in the gastropod llyanassa

495
Development 113, 495-501 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Fate maps of the first quartet micromeres in the gastropod llyanassa
obsoleta
JOANN RENDER
Department of Cell and Structural Biology, University of Illinois, 505 S. Goodwin Ave., Urbana, IL 61801, USA and
Marine Biological Laboratory, Woods Hole, MA 02543, USA
Summary
Cell fate specification in the gastropod mollusc llyanassa
obsoleta involves both cell autonomous and inductive
mechanisms, which depend on determinants localized
first in the polar lobe and then in the D quadrant of the
embryo. A complete cell lineage is lacking for this
embryo and is essential for a critical interpretation of
previous experimental results and an analysis of the
mechanisms at the molecular level.
Lineages of the first quartet micromeres were followed
using Lucifer Yellow dextran as a tracer. The tracer was
injected into individual first quartet micromeres using
iontophoresis and patterns of fluorescence were analyzed
in the larva after 8 days of development. Fluorescence
was limited to head structures, including eyes, tentacles
and velum. Structures on the left side were derived from
la and Id micromeres; la gave rise to the left eye,
including the lens. Right side structures were derived
from the lc micromere and lb contributed to the apical
plate between the eyes and symmetrically to both sides of
the velum. First quartet lineage data are compared with
results from previous cell ablation experiments and with
lineage data from other species.
Introduction
micromeres (2a-2d) arise sinistrally and third quartet
micromeres (3a-3d) arise dextrally, preceding the
production first of the 4d (mesentoblast) cell, and then
the other fourth quartet micromeres (4a-4c). Gastrulation is by epibole and is completed by the end of the
second day (Tomlinson, 1987). Organogenesis follows
for 5-6 days, resulting in a bilaterally symmetric veliger
larva (Fig. 2) with approximately 1200-1400 cells
(Collier, 1976). Polar lobe determinants in the D
macromere are essential between fifth and sixth
cleavage in the specification of cell fates, both
autonomously in derivatives of the D cell and inductively on derivatives of the A, B and C cells (Clement,
1962, 1967, 1986a, 19866). As an example of the latter,
when the polar lobe is removed during early cleavage,
embryos fail to form eyes, which are derived from A
and C cells. Both mechanisms seem to be temporally
and spatially coincident in this embryo.
The analysis of autonomous and inductive mechanisms of cell fate specification in /. obsoleta would benefit
from cell lineage data. Currently this information can
only be approximated by using lineage data from
another prosobranch gastropod, Crepidula fornicata
(Conklin, 1897), or by making inferences from cell
ablation experiments (Clement, 1967, 1986a, 19866).
Relying on cell ablation data for information on cell
lineage is dangerous because part of the embryo has
been removed, altering normal cell positions and
Embryonic cell fates can be specified autonomously via
localized morphogenetic determinants or inductively
via cell-cell interactions (Davidson, 1986). It is
common for both mechanisms to operate in a given
embryo, as has been demonstrated for the nematode
Caenorhabditis elegans. Although most cell fates are
specified autonomously in C. elegans embryos, several
examples of specification via cell-cell interactions have
been demonstrated (Greenwald, 1989). These analyses
were facilitated by knowledge of the complete cell
lineage for the embryo (Sulston et al. 1983).
Cell fates in the gastropod mollusc llyanassa obsoleta
also are specified both autonomously and inductively.
Both mechanisms are mediated by morphogenetic
determinants that are first localized in an anucleate
vegetal pole protrusion termed the polar lobe, which
forms during early cleavage. By the end of the second
cleavage, polar lobe determinants are incorporated
exclusively into the D macromere, which is larger than
the A, B and C macromeres. Three quartets of small
micromeres are produced at the animal pole of the
embryo during spiral cleavages that alternate in
direction between clockwise (dextral) and counterclockwise (sinistral). The first quartet of micromeres
(la-Id) are produced in a clockwise direction and the Id
cell is smaller than the others (Fig. 1). Second quartet
Key words: cell lineage, llyanassa, iontophoresis,
micromere, fate map, gastropod.
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J. Render
B
Fig. 1. 8-cell stage. (A) Fluorescence micrograph showing Id labeled with Lucifer Yellow dextran. Scale bar shows 100 /an.
(B) Diagram showing macromeres (A-D) and micromeres (la-Id) with Id shaded.
interactions. Such changes may lead to regulation or
may trigger greater deficiencies, especially in embryos
such as/. obsolete, in which some cell fates are specified
by inductive interactions. Some lineage information for
cells involved in the formation of the external shell was
generated by marking cell surfaces with chalk particles
(Cather, 1967); however, a complete fate map specific
to /. obsoleta is essential for the interpretation of
previous experimental and future molecular work.
External markers such as chalk or vital dyes may shift
position or fade, unlike intracellular markers such as
fluorescent, high molecular weight dextrans, which
provide unambiguous cell lineage data.
In this study, individual first quartet micromeres were
microinjected with Lucifer Yellow dextran and resulting
patterns of fluorescence were analyzed at the larval
stage 8 days later. Micromere lineages were followed
exclusively because cell ablation experiments have
indicated that the first three quartets give rise to all of
the larval ectoderm and that the mesentoblast cell (4d)
gives rise to mesodermal and endodermal structures;
the other fourth quartet micromeres and the macromeres probably contribute to the endoderm and the
yolky macromeres appear to be in part nutritive. An
added advantage to labeling individual micromeres is
that they are small and transparent and produce a
clearly discernible, discrete pattern of fluorescence at
later stages. When labeling data are compared with
previous experimental work on /. obsoleta embryos and
with lineage information from other gastropods, interesting differences emerge. For example, results from
ablation experiments in /. obsoleta indicated that the Id
micromere makes no apparent contribution to the larva
(Clement, 1967); however, when Id is labeled, it is seen
to contribute significantly to the left side of the larva.
Similarly, based on lineage information from the
related gastropod C. fornicata (Conklin, 1897), one
would expect the right eye to originate from the lb
micromere. Lineage tracing, however, demonstrates
that this structure is derived from lc in /. obsoleta.
Materials and methods
Obtaining and rearing embryos
I. obsoleta were collected near Woods Hole, Massachusetts
and were maintained in aquaria containing a mixture of
artificial and natural seawater or running natural seawater.
The aquaria were kept at room temperature and i-1 clam or
mussel was added daily as food. Egg capsules were collected
and fertilized eggs were removed from them as described by
Collier (1981). Embryos were reared individually in wells in
glass spot plates containing seawater that had been passed
through a 0.2/an filter (FSW) and supplemented with
lOOunitsml"1 penicillin and 200/igmP 1 streptomycin. Control embryos were reared at a concentration of 6 embryos/
well. Spot plates were kept in the dark at 22°C in moist
chambers. Embryos were transferred to wells of fresh
antibiotic seawater every other day.
Iontophoresis
Anionic, fixable Lucifer Yellow dextran (6%, 10,000 MT,
Molecular Probes) was filtered using a Spin-X unit (0.45/an,
Costar) and kept in aliquots at — 20 °C. Pipets for injection
were pulled from thin-walled capillaries with internal filaments (1.5 mm outside diameter, World Precision Instrument) to a tip diameter of approximately 0.2 /an using a P-87
puller (Sutter Instrument Co.). Pipets were back-filled with
dextran and 3 M KC1 was added using a syringe.
Pipet and reference electrodes were attached to a pulse
generator (designed and provided by the Zoological Laboratory, Utrecht, The Netherlands), which produces a hyperpolarizing square wave pulse. Injected current was set at
approximately 10 nA (100 mV) and the duration of each pulse
was 0.3-0.4 s. The pulse generator was attached to an
Ilyanassa cell lineage
oscilloscope (model HM-205-2, Hameg Instruments, NY) so
that changes in potential reflecting membrane penetration
could be monitored.
Injections were done under a stereomicroscope in a small
plastic Petri dish that was filled with black wax (Fisher).
Embryos were immobilized in depressions made in the wax. A
Zeiss stereomicroscope was set-up for epifluorescence using
an attachment that holds a mercury lamp (50W) and an
excitation filter (BP 450-490 nm). Barrier filters (LP 520)
were mounted on the eyepieces. Stereomicroscope fluorescence was used to monitor the progression of dextran
injection. Pulsing for a period of 1-2 min usually was
sufficient to label brightly a cell without injuring it. Most first
quartet micromeres were injected at the 8-cell stage (Fig. 1);
however, a few were injected after second quartet micromeres
had been produced. First quartet micromeres do not divide
until after the formation of second quartet micromeres. The
outcome was unaffected by the stage when injected.
Analysis
After 8 days of development larvae were anesthetized in
chlorobutanol (1 part in 2 parts FSW), and then werefixedin
10% formalin in FSW for lh at room temperature or
overnight at 4°C. Following fixation, larvae were rinsed and
mounted in deionized water under a cover slip supported with
a thin layer of clay on all sides for viewing on the compound
microscope using epifluorescence. Larvae were rolled around
during analysis, but were oriented with the velum up for
photography. Neofluar objectives (Zeiss) were used and
images were recorded on Tri-X film (Kodak, 400 ASA) using
a combination of epifluorescence and bright-field illumination. A more selective barrier filter (BP 515-565 nm) was
used during compound microscope analysis to reduce
background fluorescence.
Results
/. obsoleta embryonic cells have proven difficult to
inject using traditional, pressure injection techniques.
The addition of fluid through a relatively large
micropipet tip frequently results in cell lysis. Microinjection using iontophoresis has been much more
successful in these cells. Extremely small (0.1-0.
)
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pipet tip diameters can be used and charged molecules,
rather than bulk fluid, are delivered.
Data from 35 experiments showed that success rates
and embryo survival are good following iontophoretic
microinjection. Micromeres survived penetration with a
pipet in 108 (78 %) of 139 embryos. Transfer of Lucifer
Yellow dextran, as seen using stereomicroscope epifluorescence, was successful in 94 (68%) of these
embryos. Of the 94 embryos that developed to the
larval stage and were examined at 8 days, 72 (77%)
were perfectly normal.
Successfully injected embryos were included in the
analysis only if they developed normally and if
fluorescence was restricted to the injected micromere.
If injections were done too soon after a cell division,
both daughter cells became fluorescent as the dextran
passed through the remaining cytoplasmic bridge.
Control, non-injected, embryos were reared as small
groups in wells. Data from experiments in which half or
more of the control embryos developed abnormally
were not included in the analysis. In 45 experiments, a
total of 254 of the 271 control embryos (94%)
developed completely normally.
Patterns of fluorescence following labeling of first
quartet micromeres were remarkably consistent and
were restricted to the head and velum, which is a
bilateral, ciliated swimming structure (Table 1, Fig. 2).
The analysis of labeled larvae was aided by Tomlinson's
(1987) SEM description of morphological changes in /.
obsoleta embryos during days 4-7 of development. A
medial apical band of cilia is present, which divides the
prototrochal (anterior) area into left and right halves.
The dorsal part of this band appears to give rise to the
head vesicle, which is less prominent in /. obsoleta than
in many of the studied gastropod species. A part of the
band ventral to this gives rise to the apical plate, a
group of cells separating the left and right cephalic
plates, which form parts of the left and right velum;
eyes and tentacles arise from the bilateral cephalic
plates. The ventral-most part of the median apical band
extends laterally to form the ventral edges of the velum.
Table 1. Fluorescent areas in larvae
Cell
labeled
la
Ib
lc
Left
eye
19
0
1
_
_
-
Left
velum
20
0
0
_
_
Left
dorsal
edge
velum
16
0
0
_
_
-
Apical
plate
Ventral
edges
velum
Near
both
eyes
14
4
2
22
0
21
It
19
19
i
Id
_
0
22
2
_
23
1
0
•Unable to determine
t Lateral edges fluorescent
tOne of these near right eye only
§ Also fluorescence under left eye
_
0
24
0
l
0
18
6
0
Right
eye
0
20
0
0
Right
velum
-
20
0
0
Right
dorsal
edge
velum
Right
tentacle
15
0
0
15
0
2
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/. Render
dorsal
right
left
ventral
Fig. 2. Diagram of a veliger larva, viewed anteriorly.
v, velum; t, tentacle; sh, shell; 1, lens; e, eye; s, statocysts;
f, foot.
Fluorescent Patterns
la
Injection of Lucifer Yellow dextran into 20 la micromeres gave a consistent pattern on the left side of the
larva (Table 1). Fluorescence was found in the left half
of the velum (left cephalic plate) in all larvae and
extended into the center (apical plate) in 14 (78%) of
the larvae. The region containing the left eye, visible as
a pigmented spot, was brightly fluorescent. The eye
consists of a pigmented cup and a transparent lens. To
verify that fluorescence in the area actually included the
eye, the lens of the left eye was examined closely in 10
larvae and was found to be labeled in 9 of these (Fig. 3);
the remaining case was ambiguous. The dorsal (upper)
left edge of the velum always was labeled; however, this
was not noted in earlier experiments since the level of
fluorescence in this area was often lower than in other
parts of the pattern (note that only 16 embryos were
scored for this feature, as shown in Table 1). The use of
Neofluar objectives increased the signal from this area.
In one larva there was somefluorescencealong the right
lateral edge of the mantle. The mantle tissue is
underneath the shell and secretes shell material.
lb
The lb micromere was filled with Lucifer Yellow
dextran in 22 embryos. The fluorescence pattern in
8-day larvae was extensive and symmetrical, centered
Fig. 3. Patterns of fluorescence in 8-day larvae. (A) la was injected at the 8-cell stage. Lens of left eye is labeled (arrow).
(B) lb was injected. (C) lc was injected. Lens of right eye is labeled (arrow). (D) Id was injected. Scale bars show 100 jon;
B is at a different magnification, rv, right velum; lv, left velum; sh, shell; m, mantle edge (right).
Ilyanassa cell lineage
on the apical plate area between the eyes (Fig. 3). The
ventral (lower) edges of the velum were labeled and
the pattern extended up to, but appeared not to cross,
the lower boundary of the la edge pattern. In one
larva, the edge fluorescence was more lateral (Table 1)
but was still symmetrical. In 2 larvae, there was some
fluorescence along the left lateral edge of the mantle.
Patterns of fluorescence were found on the velum under
each eye and when closely examined, the lenses were
unlabeled in 8 of 9 larvae. In the remaining larva, it was
not possible to determine whether the lenses were
fluorescent. In one larva, a fluorescent patch was found
under the right but not the left eye. In another larva,
fluorescence was below the left eye, but covered the
right eye, although it is unknown whether the right lens
was fluorescent.
lc
A fluorescent pattern on the right side was observed in
20 larvae following injection of the lc micromere
(Table 1). This pattern was very similar to the la
pattern seen on the left side of the larva, but it seemed a
bit more extensive. Fluorescence extended from the
apical plate to the right velum and the labeled region
contained the right eye in all larvae. When the right lens
was carefully examined in 9 larvae, it was fluorescent in
6 cases (Fig. 3), while in the other 3 cases it was
impossible to determine. The dorsal right edge of the
velum was labeled, and as in larvae from la-injected
embryos, this part of the pattern was overlooked in
earlier experiments (only 15 embryos were scored, as
shown in Table 1). As with the la pattern, the area
labeled along the dorsal right edge of the velum
extended down to, but appeared not to cross, the upper
boundary of the lb edge pattern (Fig. 3). The right
tentacle, which unlike the left tentacle is present in
8-day larvae (Fig. 2), was labeled in 15 out of the 17
larvae that were closely examined; the remaining 2
cases were ambiguous. Other structures, often on the
right side, were labeled in a few larvae. The right half of
the foot and the right statocyst, a balance organ in the
foot, were fluorescent in 2 cases, while in another case
the lower edge of the foot on the right side was labeled.
The dorsal mantle edge displayed some fluorescence in
2 larvae and the right and ventral edges of the mantle
were labeled faintly in another larva.
Id
Fluorescent patterns were analyzed in 24 larvae
following injection of the Id micromere. The pattern
consistently observed was on the left side of the larva
and overlapped the la pattern (Table 1, Fig. 3).
Fluorescence was found in the left velum in 23 (96 %) of
the larvae and extended into the apical plate in 18
(75 %). In one larva, most of the fluorescence coalesced
behind the velum, with 2 lines of fluorescence extending
from the velum toward the rest of the larva. In the
typical pattern, fluorescence was near, usually under,
the left eye and, unlike the la pattern, never included
the lens (13 larvae examined). Another difference
between the la and Id patterns was that the dorsal left
499
edge of the velum was not labeled following Id injection
(Fig. 3). The mantle edge was labeled in 14 of the Id
larvae. In 9 larvae, the right lateral mantle edge
displayed some fluorescence; 3 of these also had labeled
ventral edges and one had a labeled dorsal edge, all on
the right side (Fig. 3). The ventral mantle edge on both
sides was dimly fluorescent in 3 larvae and the entire
mantle edge was labeled in 2 larvae.
Discussion
Fluorescent patterns in /. obsoleta larvae correspond
well to the positions of first quartet derivatives in the
embryo. The four quadrants of the early embryo, A-D,
give rise to a bilaterally symmetric veliger larva; D and
B quadrants form dorsal and ventral areas and A and C
quadrants form left and right sides, respectively.
Mirror-image patterns from la and lc derivatives arise
on the left and right sides of the velum. Derivatives of
lb are arranged bilaterally on the ventral side of the
velum. Similarly one might expect Id derivatives to be
bilaterally distributed on the dorsal side of the velum;
however, the Id pattern is shifted toward the left side.
This may be due to a relatively greater spiral
displacement of Id to the left as it is produced from the
large D macromere.
The slight variability observed in the patterns of
fluorescence is likely due to the death of micromere
derivatives. This could go undetected in cases where
there are no obvious defects in larval morphology when
certain cells are missing, as might be expected for some
lb and Id derivatives. Fluorescent dextrans may be
released from the injured cell(s) and become dispersed
or coalesce to form aggregates in the embryo. Foot and
statocyst fluorescence in a few lc larvae is probably due
to the undetected passage of dextrans from lc to the C
macromere following injection. Such patterns of labeling are expected for the 2c and 3c micromeres, which
are produced later by the C cell (Clement, 1967;
unpublished data).
Ablation experiments in /. obsoleta and in another
prosobranch gastropod, Bithynia tentaculata, in which
the entire first quartet of micromeres was killed with a
glass needle at the 8-cell stage, resulted in larvae lacking
head structures including eyes, tentacles and velum
(van Dam and Verdonk, 1982; Sweet and Boyer, 1990).
Although lineage data are consistent with these results,
significant differences exist when data from the ablation
of individual first quartet micromeres are considered.
The ablation of Id in /. obsoleta appeared to have no
effect on larval development in the three cases reported
by Clement (1967), although ablation of Id in B.
tentaculata (van Dam and Verdonk, 1982) resulted in
embryos that had a reduction in the left side of the head
and that often lacked the left eye. These defects are
similar to those observed following ablation of la and
are consistent with lineage information from /. obsoleta. Although Id normally does not contribute to the
left eye in /. obsoleta, its descendants contribute to the
left velum (Fig. 3). Since the fates of la and Id overlap
500
J. Render
(parts of the apical plate and left velum), normal larvae
may have resulted following ablation of Id in /. obsoleta
because of regulation by la descendants. It is also
possible that the region of velum contributed by Id, if
missing, would be difficult to detect. The very small
sample size (3 cases) makes the ablation results difficult
to interpret with confidence.
A low level of fluorescence was observed along the
mantle edge in a few larvae from la-, lb- or lc-injected
embryos and in many larvae from Id-injected embryos.
Cell deletion and marking experiments have shown that
the 2c and 2d micromeres give rise to external shellforming tissue, including the mantle edge (Clement,
1967; Cather, 1967). There was no indication that the
deletion of any first quartet micromeres resulted in shell
abnormalities (Clement, 1967). First quartet micromeres, notably Id, may play a small role in normal shell
formation that is not detected when these cells are
deleted.
In other cases, data from ablation experiments and
lineage analysis were in agreement. When the la
micromere was ablated in /. obsoleta at the 8-cell stage,
resulting 7-day-old larvae lacked the left eye and left
dorsal part of the velum, but appeared quite normal in
other respects (Clement, 1967). Embryos with an
ablated lc micromere gave rise to larvae missing the
right eye and tentacle and the right dorsal part of the
velum. These results agree completely with the la and
lc lineages, which contribute to the left eye and dorsal
left velum and right eye, tentacle and right, dorsal
velum, respectively (Fig. 3). Results from the ablation
of lb also agree well with the lineage of this cell. In
larvae from embryos lacking lb, the apical plate region
between the eyes was reduced, in some cases quite
severely, resulting in the unusually close medial
placement of the eyes. Ablations of la, lb and lc
micromeres in B. tentaculata gave similar results
(Cather et al. 1976; van Dam and Verdonk, 1982).
Information from cell lineage analyses of a few other
gastropod molluscs is available and can be compared
with the /. obsoletafirstquartet data. No lineage tracers
were used in these studies, which consisted of observing
cell divisions and analyzing embryos that werefixedand
stained at various stages. Among existing lineage work,
Conklin's (1897) analysis of Crepidula fornicata seems
most useful for comparison, since both /. obsoleta and
C. fornicata are marine prosobranch gastropods, both
form polar lobes during early development and both
form free-swimming veliger larvae. Conklin's assessment of the origin of head structures in C. fornicata
agrees in many respects with the lineage data generated
for /. obsoleta, although some significant differences
exist. Conklin believed that the right eye in C. fornicata
formed from descendants of lb, unlike the lc origin
demonstrated in /. obsoleta. In C. fornicata, Id made no
obvious contribution to larval structures although, in /.
obsoleta, Id contributes to the apical plate and left
velum. A reexamination of the C. fornicata lineage
using fluorescent tracers may help resolve this inconsistency.
No lineage data exist for B. tentaculata; however, one
would expect cell fates to be similar to those in /.
obsoleta. If Id were labeled with a fluorescent dextran
in a B. tentaculata embryo, it seems likely that
fluorescence would be found in the left cephalic plate
and, unlike in /. obsoleta, would include the left eye.
Discrepancies between cell ablation and blastomere
isolation (partial embryo) experiments in /. obsoleta
may be better understood in light of lineage data. When
CD half embryos were isolated at the 2-cell stage,
30-40% of the resulting larvae formed two eyes
(Clement, 1956; Render, 1989). In larvae from BD half
embryos that were created at the 4-cell stage, 11%
formed two eyes (McCain and Cather, 1989) and some
D partial embryos gave rise to larvae that formed one
eye (Clement, 1956). This seems to contradict results
from micromere ablation experiments (Clement, 1967)
and lineage analysis, which together demonstrate that
eyes are derived from the la and lc micromeres.
Lineage data also show that Id contributes to the left
velum near the eye and lb contributes to both right and
left velum near the eyes. It seems that regulation may
occur when the A quadrant or A and C quadrants are
removed before first quartet micromeres are given off
at third cleavage. In these situations, Id may fill in for
la in the formation of the left eye and lb may fill in for
lc in the formation of the right eye. Interestingly, one
lb larva in this study appeared to have fluorescence
over the right eye (Table 1). By the third cleavage,
regulation does not occur following removal of the la or
lc micromere; the fates of these cells seem to have
become specified by this stage. The fates of la and lc do
not seem to be fully determined until a later stage,
however. By ablating the D macromere at successively
later stages, Clement (1962) demonstrated that inductive influences from this cell, which contains morphogenetic determinants from the polar lobe, are required
until between the fifth and sixth cleavages. Before this
stage ablation of the D macromere results in larvae that
lack eyes and many other lobe-dependent structures.
Cell lineage analysis in /. obsoleta provides an
essential baseline of information on normal development to which data from experimental manipulations
can be compared. Lineage analysis will be extended to
other quartets of micromeres in /. obsoleta to complete
the data on normal development. Combining lineage
tracing with experimental manipulations, such as cell
ablation or the redistribution of polar lobe determinants (Render, 1989), should prove useful in studying
mechanisms of cell fate specification in /. obsoleta. For
example, experiments are in progress in which the
micromeres (lc or Id) of a CD embryo are labeled to
determine the origin of the extra eye that appears in
many resulting larvae. Is the Id lineage converted to a
la lineage in such embryos as might be expected? If so,
how is the conversion accomplished? Cell lineage
information will also be valuable in analyzing these
mechanisms at the molecular level.
I am grateful to Florenci Serras and Jo van den Biggelaar
for instruction in iontophoresis and for their gracious
hospitality during my visit to Utrecht. Barbara Boyer and
Ilyanassa cell lineage
Richard Sanger provided technical advice and generously
loaned equipment. James Cather and Jonathan Henry and
two anonymous reviewers offered helpful critiques of the
manuscript. This work was supported by a Steps Toward
Independence Summer Fellowship from the Marine Biological Laboratory and by NIH grants HD23104 and HD27328.
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(Accepted 27 June 1991)