J. Embryol. exp. Morph. 86, 1-17 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
Formation of the notochord in living ascidian
embryos
DAVID M. MIYAMOTO
Department of Biology, Seton Hall University, South Orange, NJ 07079, U.S.A.
Marine Biological Laboratory, Woods Hole, MA 02543
AND ROBERT J. CROWTHER
Boston University Marine Program, Marine Biological Laboratory, Woods Hole,
MA 02543, U.S.A.
SUMMARY
The dynamic behaviour of cells during formation of the notochord in the ascidian, Ciona
intestinalis, was examined by means of Differential Interference Contrast (DIC) microscopy and
time-lapse videorecording. The initial rudiment is formed in part as a consequence of the pattern
of mitotic divisions as the blastopore shifts posteriorly. Vertical and horizontal rearrangements
produce an elongate rod of disc-shaped cells stacked end to end. Further elongation is accompanied by a cell shape change. Some cell growth or swelling is indicated to occur later in
development, but this growth appears to contribute mostly to an increase in the diameter, and
only insignificantly to the length of the notochord. Intracellular vacuoles that appear around 13 h
after fertilization increase in size and fuse at about 16 h to form intercellular ones. These in turn
merge to form the central matrix core of the notochord at around 18 to 20 h. As the notochord
elongates and cells change in shape, the basal surfaces bleb actively. This surface activity may be
related to formation of the perinotochordal sheath.
INTRODUCTION
Several cell behaviours considered to be important in forming embryonic structures are displayed during formation of the notochord. Cell proliferation, growth,
migration, rearrangement, shape changes, and alteration of the extracellular environment occur as the notochord is transformed from a loose plate or mass of cells
into an elongated rod surrounded by a sheath of fibrous material. Notochord formation is thus an ideal event to examine the roles that these behaviours play in how
this and other embryonic structures are formed and how the embryo is shaped.
There have been a number of descriptive studies on notochord morphogenesis
in a variety of different chordates (Mookerjee, Deuchar & Waddington, 1952;
Lesson & Lesson, 1958; Bancroft & Bellairs, 1970; Ruggeri, 1972; Malacinski &
Youn, 1982). Notochord formation has not been examined directly in these organisms, however, because of the size and opacity of their embryos. Studies of internal
Key words: Ascidian embryo, cell rearrangements, cell shape changes, intracellular vacuoles,
video time lapse, notochord.
2
DAVID M. MIYAMOTO
cell behaviour in intact, living embryos are possible in a number of species of
simple ascidians since they are relatively transparent. Prior studies on ascidian
notochord formation have characterized the lineage of presumptive notochord
cells by endogenous (Conklin, 1905; Reverberi, 1971) or applied cell markers
(Ortolani, 1955; Nishida & Satoh, 1983), cellular interactions and the determinative factors responsible for specifying notochord differentiation (Reverberi,
1971; Whittaker, 1979), structure of the notochord in the larva (Welsch & Storch,
1969; Katz, 1983), changes of the notochord during metamorphosis and tail
resorption (Cloney, 1969; 1982), and involvement in neural plate induction (Rose,
1939; Reverberi, Ortolani & Farinella-Ferruzza, 1960; Reverberi, 1971). Early
descriptions of notochord formation (Conklin, 1905; Berrill, 1955) do not examine
suggested morphogenetic mechanisms in a critical manner whereas ultrastructural
studies of Cloney (1964) and Mancuso & Dolcemascolo (1977) do not examine
directly any of its dynamic aspects. This study takes advantage of the relative
transparency of embryos of the ascidian, Ciona intestinalis, the ability of Differential Interference Contrast (DIC) microscopy to optically section and visualize
details in thick specimens, and the improved contrast resulting from current videotechnology (Allen, Allen & Travis, 1981), to follow presumptive cells from midgastrulation through tadpole formation and describe their dynamic behaviour as
they form the notochord.
MATERIALS AND METHODS
Culturing of the embryo
Gametes of Ciona intestinalis were obtained from mature adults, fertilized, dechorionated, and
cultured according to procedures described by Crowther & Whittaker (1983). Embryos to be
studied were placed in a perfusion chamber constructed out of microscope slides, coverglasses,
No. 18 syringe needles, intramedic tubing and epoxy cement. The chamber was thin enough
(1—1-2 mm) to use oil immersion and other high numerical aperature objectives while maintaining
a temperature of about 19 °C and a continuous flow of fresh sea water past the embryo. Newly
fertilized embryos placed in this chamber developed normally to the time of hatching (18-20 h).
Development times reported are the actual age of the embryo since fertilization at the culture
temperature. Development occurs at the rate reported by Whittaker (1979) for 18 °C. Because
the tail begins to twitch around 13 to 15 h of development, embryos were anaesthetized by
perfusing 0 • 1 mM-nicotine through the chamber beginning at 12 h postfertilization. This treatment
had no adverse effects on development whereas other anaesthetics tried (MS-222, Chlorotone)
stopped both tail elongation and further development of the notochord.
DICIVideomicroscopy
A Leitz microscope equipped with Nomarski DIC optics was used to make the recordings. A
Dage Model 65 Newvicon camera connected to a time-lapse videorecorder (either a Sony model
TVO-9000 (finch tape) or a Panasonic model NV-8050 (Hnch tape)) with time-date generator,
and high-resolution videomonitors comprised the videosystem. Tapes were recorded at speeds
ranging from 1/12 to 1/36 actual speed. Photographs were taken of the monitor screen with a
35 mm camera as described by Allen et al. (1981). Scan lines were eliminated in the photographs
by using a Ronchi grating, 50 cycles/inch (Rolyn Optics, Covina, CA) either during the making
of the negative or during printing. The mapping of cell rearrangement as well as the measurements of tail length, cell diameter, and cell length were done from tracings of the monitor screen
Notochord formation in ascidians
3
of selected time points using transparent acetate sheets. Cell volume was calculated by assuming
cells were perfect cylinders of constant diameter.
The emphasis of this study was on observing the dynamic behaviour of cells, events that can
only be seen when they are speeded up during playback of the videorecordings. Events that
occurred in a relatively brief moment of time could be captured and observed using various
playback features of the recorder (different speeds, forward and reverse playback, stop field and
one shot advance). Some of the changes are such that even the most patient of still photographers
would have missed them. Other advantages of the technique are (1) developmental changes in
the same cells could be followed continuously through time; (2) constant refocusing was unnecessary since videorecording is vibration free; (3) only low levels of illumination are required,
reducing the amount of heat generated that can kill the embryo; (4) a record of time is provided
by the time-date generator; and (5) results can be viewed immediately upon completion of
taping. The major problem with the technique is that the videorecorder has a horizontal
resolution of only 300+ lines. The resolution of the camera and monitor is 700 lines. Stopping on
a particular 'frame' of the tape in order to photograph the screen resulted in further loss of
definition as only one set of scan lines is displayed.
Histology
In vivo studies were complemented by histological studies to examine some of the changes
difficult to see with the video. Styela clava, another species of ascidian, was used to take advantage
of its distinctive differences in pigmentation and cytoplasm that mark cells of different presumptive fates (Conklin, 1905). Gonads from two different adults were minced together to fertilize the
eggs and debris was filtered out using nylon mesh cloth. Various stages were fixed with 2%
glutaraldehyde in sea water, embedded in JB-4 resin (Polysciences), and serially sectioned at
4 jum using glass knives on a AO rotary microtome. Measurements and reconstructions were done
from drawings of complete sets of serial sections made using a projection microscope.
Electron microscopy
Ciona embryos at selected stages of development werefixedin 2-5 % glutaraldehyde in a 0-2 Mphosphate buffer (pH7-2), containing 0-34M-NaCl for 60mins, and postfixed in 2% osmium
tetroxide in 1-25 % bicarbonate buffer (pH 7-2) for 15 mins. After a buffer wash, they were then
dehydrated through an ethanol series, cleared with propylene oxide, and embedded in an Embed
812-Araldite mixture (Mollenhauer, 1964) at 60 °C in flat embedding moulds. Thin sections were
cut with a diamond knife on a Sorvall MT-2 B ultramicrotorne and stained at room temperature
with alcoholic uranyl acetate (7-5 % in 50 % ethanol) for 3 to 5 mins and aqueous lead citrate
(Reynolds, 1963) for 3 mins. Preparations were examined and photographed in a Zeiss IOC
transmission electron microscope at 80 kV.
RESULTS
Formation of the notochord rudiment
Early changes during notochord formation are as described by Conklin (1905).
At the start of gastrulation presumptive notochord cells are arranged in an arc that
lies anterior and lateral to the endoderm (Fig. 1A). As invagination begins, these
cells elongate, extending radially from the anterior margin of the blastopore. As
blastopore closure occurs, cell division converts the initial single arc of cells into two
(Fig. IB) and then three tiers of cells.
Examination of the time-lapse videorecordings shows closure of the blastopore
to be due mostly to the posterior movement of the anterior margin of notochord and
overlying neural plate cells between flanking bands of muscle and mesenchyme
(Fig. IB). Consequently the descendants of the central cells of the arc (Fig. 1A) that
DAVID M. MIYAMOTO
Fig. 1
Notochord formation in ascidians
5
are still attached to the blastopore margin become the posterior-most cells of the
rudiment formed by the notochord cell lineages established by Conklin (1905) and
Ortolani (1955) (Figs IB, 1C). However, the blastopore does not end up at the very
posterior tip of the embryo but rather stops anterior to it (Fig. ID). As the blastopore moves posteriorly and becomes smaller, notochord cells that line it become
wedge shaped, but their constricted apical surfaces do not show any activity such
as blebbing. Cell contours remain smooth and no surface extensions are seen that
can be interpreted to be motile.
Rearrangement of notochord cells
As the neural tube is formed, the width of the notochord rudiment decreases as
it elongates. This change is accompanied by cell rearrangement (Fig. 2). Cells
exchange neighbours, moving toward the midline of the rudiment and causing
longitudinal separation and displacement. As cells elongate across the axis of the
rudiment they become wedge or spindle shaped with tapered ends that extend
between neighbouring cells. Once they reach the opposite side they become disc
shaped, appearing rectangular in the recordings (Figs IF, 2H). The resulting
sequence of stacked cells appears to be random. Cells that start from the same side
Fig. 1. Notochord rudiment (A-D) and vacuole (E-F) formation. All times in this and
other figures are since fertilization.
(A) Midgastrula-stage embryo (4-5 h). Notochord cells (n) form an arc that lies anterior to presumptive endoderm (e); m, muscle and mesenchyme. Solid arrow marks the
same medial cell similarly marked in (B, C, D). Scale bar = 20/u,m.
(B) Same embryo, 5-5 h. Notochord cells have divided to produce two tiers of cells, a
marginal row (nl) and an internal row (n2). Anterior blastopore lip is advancing posteriorly (large open arrow) between the two flanking bands of presumptive muscle/
mesenchyme (m). Posterior muscle cells (pm) have come into contact along the midline.
(C) Blastopore closure nearly completed (6-5 h). Descendant of the medial cell (solid
arrow) is now one of the posterior-most cells of the rudiment. Only few muscle cells (m)
are still visible in this plane of focus as most have invaginated inward as the ectoderm
begins to curl upwards.
(D) Blastopore closure complete (7 h). The cell being followed (solid arrow) has moved
out of the plane of focus into the closed blastopore. The blastopore becomes included
within the embryo as posterior ectoderm shifts anteriorly and mediad (large open
arrows) and the neural tube is formed.
(E) Cell rearrangement almost complete (10-5 h). For the location and orientation of
the sequence to follow (1E-F, 3 A-G) see Fig. 3H. Scale bar = 10 pm. A number of cells
have not yet completed interdigitation and are still wedge shaped with tapered margins
(solid arrow) that in the videorecordings appear to undulate (compare with Figs 2G, H).
ec, ectoderm; e, endodermal strand.
(F) Cell rearrangement complete and notochord cells (n) are disc shaped and arranged
in a single row (11 h).
(G) Vacuoles (v) become visible around 13 h. Diameter of the notochord has decreased
as cells have increased in their length.
(H) Intracellular vacuoles have increased in size and are found as adjoining pairs
(14-5 h). Surfaces of notochord cells are actively blebbing (large open arrow). Solid
arrows indicate the same vacuoles seen in Fig. 3. Note the change in cell diameter and
length between (F) and (H).
DAVID M. MIYAMOTO
e
D
Fig. 2. Mapping of cell rearrangements. Individual cells (a-e, t-z, *) were followed in
the videorecording and their displacements and shape changes mapped on acetate
sheets. Large open arrow indicates the direction of the posterior pole, the direction of
tail and notochord elongation. Dashed lines indicate out-of-focus regions. Scale bar in
A = 10/u,m. Cells from the two sides (a-e, t-z) move toward the midline, come into
contact (B) and interdigitate (C, D, E). Neighbouring cells lying in the middle of the
rudiment (*) are separated and displaced along the axis of the rudiment as this occurs
(A-D). During the later stages of interdigitation (F, G, H), cells pulsate and move back
and forth parallel to the direction of their movement. Cells from one side (a, b) may
alternate in position with those from the other side (x, y) upon completion of interdigitation. Alternatively, cells from the same side remain as neighbours (b, c). Not all of the
cells could be positively mapped from the beginning to the end of the sequence because
the tail curled out of the plane of focus as it elongated (C, D, E, F). Changes in embryo
position (B, C, D) or refocusing of the camera (E, F) are responsible for some of the
changes in cell size and shape diagrammed. Development times = (A) 9 h 5 min, (B) 9 h
29min, (C) 9 h 34min, (D) 9 h 53 min, (E) lOhOlmin, (F) 10h 49 min, (G) 10h 56 min,
(H) 11 h 04 min.
of the primordium may or may not end up as neighbours once interdigitation is
complete (Fig. 2H), an observation that agrees with the results of Nishida & Satoh
(1983). Vertical rearrangements also occur. Beginning as a single layer (Fig. 3A),
the notochord becomes a bilayer (Fig. 3B) and then becomes circular to oval in
cross section (Fig. 3C).
Notochord formation in ascidians
Fig. 3. Transverse sections of Styela clava. Unstippled cells, notochord; dense small
stippling, neural cells; sparse small stippling, ectoderm/epidermis; large stippling,
mesenchyme/muscle; open stippling, endoderm. Bar = 50/urn.
(A) Gastrula-stage embryo that is equivalent to a 6-6-5 h Ciona embryo. Notochord
cells are organized as a monolayer that reconstruction shows to be three rows in length
and six to eight cells across.
(B) Late-gastrula-stage/early-neurula-stage blastopore shaped like a small slit,
equivalent to 6-5-7 h Ciona embryo. Notochord cells are no longer organized as a
monolayer and are beginning to show vertical rearrangements. Neural plate is folding
to form a tube.
(C) Tail-bud-stage embryo, equivalent to 8-9 h Ciona embryo. Neural tube has formed. Notochord cells are organized into an ellipsoid mass that is circular to oval in cross
section and is two cells high and two cells across.
The interdigitating movement of cells appears to be due to the penetration between neighbouring cells of the tapered cell projection (Figs IE, 2) as reported by
Cloney (1964). During the early stages of interdigitation (Figs 2A-D) the image is
not sharp enough to see the cellular activity associated with interdigitation. As the
diameter of the notochord and tail decreases and the image becomes clearer, careful study of the videotapes show the cells to be pulsating as rearrangement takes
place (Figs 2E-H). The entire surface of the notochord cell adjacent to the
perinotochordal sheath bulges in and out in a rhythmic fashion. Cells and cell
contents exhibit back-and-forth movements that run parallel to the direction of cell
translocation. In the best of the recordings, the leading, tapered extensions appear
to show minute dilations and attenuations while at other times they have an undulating or wave-like quality.
Vacuole formation
By the time notochord cells have completed their rearrangement, the embryo is
10-11 h old and is comma shaped (Fig. 4H). Vacuoles begin to appear in some of
the cells around 13 h (Figs 1G, 5A, 5B). At first not all cells have these vacuoles
(Figs 1G, 5A) and they appear and disappear from view. By 14-14-5 h, however,
they are larger and stable, arranged in pairs to either side of a separating boundary
(Fig. 1H). These boundaries remain distinct as the vacuoles increase in size (Figs
DAVID M. MIYAMOTO
\
JiH
Fig. 4 for legend see page 10
Notochord formation in ascidians
Fig. 5 for legend see page 10
10
DAVID M. MIYAMOTO
1H, 4A). The vacuoles are at first irregular in shape (Fig. 5A, 5B). Their behaviour
is very dynamic as they are constantly bulging inward and outward in the recordings. As they become larger they become ellipsoid or spherical (Fig. 4A) and their
activity declines in intensity.
At about 16 h after fertilization the boundary separating adjoining vacuole pairs
becomes less distinct (Figs 4B,C) and disappears as they fuse to form one larger
vacuole (Fig. 4D). In shape and position these fused vacuoles correspond to the
intercellular vacuoles described by Cloney (1964). Vacuolar growth continues (Fig.
4E,F) as the cytoplasm of notochord cells become less granular. At the time that
hatching would normally occur (18-19h after fertilization), cells shift toward the
periphery of the notochord and vacuoles merge (Figs 4F,G). At this point the tail
Fig. 4. Fusion of adjoining vacuole pairs (A-D) and their merger to form the central
matrix core (E-G). Continuation of sequence Fig. 1E-H with the same orientation and
magnification (scale bar = 10|u,m). Solid arrow(s) mark the same pair of vacuoles seen
in Fig. 1H.
(A) Adjoining vacuole pairs have increased in size but are still separated by a distinct
boundary (15-75 h).
(B) Five minutes later the constriction widens.
(C) Another five minutes and the dividing line is no longer clearly visible.
(D) Twenty-five minutes after the beginning (3A) the two vacuoles are one.
(E) This rounded vacuole increases in size (17 h).
(F) By the time that hatching normally occurs (18 h), intercellular vacuoles are very
large and the separation between them is only a faint line.
(G) Vacuole merger completed. Notochord cells shift toward the periphery as the
vacuole growth occurs. In some places the distance between matrix core and adjacent
tissues of the tail becomes small (large, open arrows).
(H) Comma-shaped embryo at the beginning (10 h) of the recorded sequence, body
towards the left, tail towards the right, dorsal side toward the top of the figure. Box
indicates general location of area that was examined in Fig. 1E-H, 3A-G. Scale
bar = 50/-on.
(I) Same embryo at the same magnification at the conclusion of recording (19-5 h).
Control (non-dechorionated embryos) have hatched. Tadpole has almost completed tail
elongation, but a small amount of lengthening is still taking place. Not all of the vacuoles
have merged.
Fig. 5. Transmission electron micrographs showing early vacuole formation. Ciona
intestinalis embryo 13 h. A longitudinal section through the nuclei of the notochord
cells. The anterior end of the embryo is toward the left and posterior end toward the
right of each micrograph.
(A) Notochord cells are lined up in a single row. An intracellular vacuole (outlined by
square) is present along the anterior border of one cell. Other large intracellular inclusions are found adjacent to the lateral border of the cell (enclosed by circle). Not all
of the cells have vacuoles at this time. Scale bar = 10 (xm.
(B) The cell containing the intracellular vacuole that is located along the anterior
border identified in (A) is seen more clearly. Note that membranes (arrow) of adjacent
notochord cells are in close contact and that there is no indication of accumulation of
any material between chordal cells at this time. Scale bar = 2/xm.
(C) Intracellular vesicle (ve) encircled in (A) is seen at higher magnification. The
vesicle (ve) is membrane-bound and contains faint traces of material. They are located
adjacent to the forming sheath (s) in the area that shows blebbing activity (see Fig. 1H);
y, yolk platelet. Scale bar = 0-2 /im.
Notochord formation in ascidians
11
800
E
3 600
-
400
200
10 12 14 16 18 20
Hours after fertilization
Fig. 6. Rate of tail elongation in Ciona embryos. Length of tail from where it joins the
body to its tip as a function of time after fertilization. Closed circles, open circles, and
crosses are average values for three different sets of dechorionated embryos that were
raised in the chamber used for time-lapse videorecordings. Their sample sizes range
between three and six tadpoles. Standard error bars were not included for reasons of
clarity. Open triangles represent tail length of tadpoles for which measurements of
notochord cell diameter, length, and volume are presented in Fig. 7. Tail elongation is
approximately linear and begins to level off at about 20 h (data for older tadpoles not
shown).
has almost reached its maximum length (Figs 4F, 6) and the notochord is a central
core of clear material that is surrounded by attenuated notochord cells (Fig. 4G).
Surrounding the notochord cells is a sheath of fibrillar extracellular material (Fig.
5C; Cloney, 1964; Katz, 1983).
Quantitative analysis of cell-shape changes
After gastrulation is complete, tail elongation in Ciona proceeds at a constant
rate of about 1-3 /an/minute (Fig. 6). At the magnifications that were used, the tip
of the tail moves out of the field of view as it elongates. Most recordings were made
near where the tail joins the body of the developing tadpole (Fig. 4H) in order to
monitor cellular changes taking place continuously. Cell diameter decreases and
cell length increases in this area as the tail elongates (Figs. 1E-H; 7A). These
changes occur mostly during the early part of tail elongation and begin to decline
in extent at about 14h (Fig. 7A). After adjoining vacuole pairs have fused (16h)
there is no indication of any further longitudinal movement or cell lengthening in
this region (Figs. 4D-G; 7A). Since the rest of the tail is still elongating, it appears
that cell shape changes are completed first in anterior part of the notochord before
they are in the posterior part.
There is a gradual rise in the estimated cell volume up until about 14 h (Fig. 7B).
Comparing the data for this period (10-14 h) by means of analysis of variance
resulted in F-values that were not significant at a 95 % confidence level. The interpretation of this result is that growth is at best a minor component and notochord
12
DAVID M. MIYAMOTO
A
25
Diameter
20
6-0
15
^5-0
o
40
10
Length
3-0
10
12
14
16
18
Hours after fertilization
20
10
12
14
16
18
Hours after fertilization
20
Fig. 7. (A) Changes in the diameter and length of notochord cells. Measurements
taken from a single recording as according to materials and methods. Data (not presented)
from two other recordings show similar values and trends. Bars are 99% confidence
intervals, sample sizes ranged from three to six measurements per average, curves were
drawn by eye. Arrows indicate the following events: a, cell arrangement complete, cell
organized as a single row (shortly after Fig. IE); b, vacuoles become visible in the
recordings (see Figs. 1G, 4); c, fusion of intracellular vacuoles to form intercellular ones
(see Figs. 3A-D).
(B) Changes in notochord cell volume. Volume was calculated from same data used for
(A) by assuming the cells are perfect cylinders. Bars = 99% confidence limits. Single
classification analysis of variance indicates that values between 10 and 14-5 h are not
different at a significance level of 95%. There is an increase in cell volume after 14-5 h.
During this period the 99% confidence limit bars are quite large, indicating a great deal
of variability in volume estimates. Comparison with (Fig. 6A) shows that the time of
greatest volume increase corresponds to the time that cell length is increasing the least.
elongation during this period is principally due to changes in cell shape. After 14 h the
average cell volume rises as intracellular vacuoles grow in size (Fig. 7B). This change
is not uniform, however, since the amount of variability in cell volume is greater for
this period than the one preceding (Fig. 7B). Comparison of the length, diameter,
and volume measurements shows that the increase in volume is principally the
consequence of an increase in notochord diameter after 14 h (Fig. 7A). More importantly, the major component of apparent growth or swelling that occurs during
notochord formation happens after cells have completed most of their lengthening.
Cell blebbing during cell elongation
Cell surfaces adjacent to the other surrounding tissues of the tail being to bleb
extensively as notochord cells begin to elongate (Fig. 1H). Blebbing occurs only
after interdigitation is complete and is therefore not a component of cell movement
involved in interdigitation. Transmission electron micrographs show that during
Notochord formation in ascidians
13
this period there are membrane-enclosed vesicles adjacent to these surfaces that
contain fibrillar and amorphous material (Fig. 5C; Crowther, unpublished data;
Cloney, 1964; Mancuso, 1973). Cloney (1964), and Mancuso & Dolcemascolo
(1977) suggest that this period is one of very active secretion by notochord cells of
perinotochordal sheath material. The blebbing observed here, therefore, is
believed to be associated with this secretory activity.
DISCUSSION
Formation of the notochord rudiment
Chalk marks located on the anterior lip of the blastopore do not end up at the
posterior tip of the notochord but rather some distance from it (Ortolani, 1955;
Reverberi, etal. 1960). Cell lineage studies that mark early blastomeres by injecting
them with horseradish peroxidase (HRP) suggest that the most posterior notochord
cells are not products of the established notochord cell lineage, but are derived
instead from posterior blastomeres of the 8-cell stage (Nishida & Satoh, 1983). The
time-lapse videostudies of morphogenetic movements corroborate that finding.
They also help in understanding Nishida & Satoh's (1983) report that HRP-stained
muscle cells appear on the side contralateral to the one injected since the strict
separation between muscle and mesenchyme bands of the left and right sides that
results from the passage of notochord rudiment between them does not occur past
the point where the blastopore stops its movement (Fig. ID), the point at which
these particular muscle cells appear to be located.
Blastopore closure is associated with apical constriction and the formation of
wedge-shaped cells in a fashion similar to that described for other examples (see
Trinkaus, 1976, 1984). Posterior movement of the margin, however, does not
appear to be due to cell motility that involves motile cell extensions (see Trinkaus,
1976, 1984). No surface activity that could be associated with active motility was
observed. Scanning electron micrographs of ascidian gastrulae also give no indications of motile activity (Satoh, 1978).
Vacuole formation
Cloney (1964) and later Mancuso & Dolcemascolo (1977) report that the large
vacuoles between cells are formed by direct accumulation of material between cells
and that these intercellular structures later merge as a consequence of cells shifting
toward the sheath as the central matrix core is formed. In contrast, Berrill (1947)
reports that the vacuole formation is intracellular and vacuoles grow 'until the ends
of adjoining cells break down and the notochord becomes essentially a long continuous cylindrical vacuole, enclosed by fused peripheral cell walls'. With video
time lapse the development of particular vacuoles in Ciona could be followed
continuously from the blastula stage until completion of tadpole formation. The
recordings clearly show that vacuoles develop initially as adjoining pairs that fuse
to form a single, larger vacuole. The interpretation of this observation is that
14
DAVID M. MIYAMOTO
vacuoles initially develop as intracellular ones and that these then fuse to form
intercellular ones. That early vacuoles are indeed intracellular could be shown by
using electron microscopy to examine embryos at the particular times indicated by
the videorecordings (Fig. 5). The thin and fragile boundary that separates vacuole
pairs later in development appears to be easily disrupted, possibly because of
osmotic stress produced during preparation for electron microscopy (Crowther,
unpublished observations). Showing it in an intact state by electron microscopy
during these stages has not been possible. Subsequent events, as observed by continuous videorecording, agree with Cloney's and not with Berrill's description.
Intercellular vacuoles continue to increase in size and later merge as cells shift
toward the periphery of the notochord.
Why do these observations differ from those of Cloney (1964)? The micrographs
presented in Cloney's (1964) paper show a very early stage in vacuole formation
where adjoining cell membranes have just begun to separate and a much later stage,
just prior to hatching, where the intercellular vacuoles are well formed. There is a 4 h
gap between the two sets of micrographs in which there is only a description of the
intermediate stages of vacuole formation that is based on observations with a dissecting microscope. It may be that Cloney missed examining closely the critical stages
indicated by continuous time-lapse recording. Another possible explanation is that
formation of the notochord is not the same for all species of ascidians. For example,
in the tunicate Dendrodoa no vacuoles or central matrix core are formed (Welsch &
Storch, 1969) whereas in a number of other species only small vacuoles are formed
that never merge to form an inner matrix core (Berrill, 1947; Cloney, 1964). In most
vertebrates mtazcellular vacuoles are formed but no matrix core is formed
(Mookerjee, etal. 1952; Lesson & Lesson, 1958; Jurand, 1962,1974; Waddington &
Perry, 1962; Bancroft & Bellairs, 1970; Ruggeri, 1972). Other species need to be
examined by time lapse in order to determine if this is the reason for the discrepancy.
Notochord cell behavior and tail elongation
Tail elongation depends on the formation of the notochord. Partial embryos,
from which all presumptive notochord cells have been deleted, form rudimentary
tails that do not elongate completely (Reverberi, et al. 1960). The small amount of
elongation that does occur is probably due to rearrangements and shape changes
of the muscle cells that are present (see Cavey & Cloney, 1974). Partial embryos
that contain presumptive notochord cells and lack muscle cells form elongate tails,
the length of which depends somewhat on the number of notochord cells present
(Reverberi, etal. 1960). The quantitative data presented in this paper indicates that
cell growth or swelling is not a major cause of elongation. Berrill's (1955, 1975)
proposal that elongation is due to the colloidal matrix imbibing water and generating a pressure that is restricted by the perinotochordal sheath to exert its force along
the axis of the embryo is not correct. Most of the elongation in a region occurs when
the vacuoles are still small and before the first indications of major growth or
swelling are apparent. The fact that not all urodele species of ascidians form
Notochord formation in ascidians
15
intercellular vacuoles or a matrix core in the tail (Berrill, 1947; Cloney, 1964) is
another argument against swelling or growth of the notochord being important in
shaping the tail.
After formation of the initial rudiment by cell division, notochord cells interdigitate and rearrange to form a rod shaped structure in which they are lined up end
to end (Conklin, 1905; Cloney, 1964). Blastomere deletion and isolation experiments show that notochord cells have a tendency to line up end to end even when
normal structural relationships with other presumptive tissues have been greatly
disrupted (Von Ubisch, 1939; Rose, 1939; Reverberi, etal. 1960; Reverberi, 1971).
These experiments suggest that the ability to rearrange is intrinsic to notochord
cells and is not the result of external forces. This implies that the mechanism of
neighbour exchanges is an active one. The videorecordings suggest two possible
mechanisms by which interdigitation may be occurring. The pulsating movements
could be indicative of forward surges of cytoplasm that 'push' the tapered extension
between neighbouring cells. New cell adhesions are formed, followed by a period
of reorganization of the cytoskeleton, and then another forward surge occurs. The
other possibility is that the undulating movements of the tapered extension
represent membrane ruffling that is modified by the geometry of the substrate
(neighbouring cells) encountered (see England & Wakely, 1979). In this case interdigitation is postulated to be due to active cell motility, since ruffling is a behaviour
associated with motility in other cells (see Trinkaus, 1976; 1984).
Cell rearrangement appears to be an important component of the shaping of
embryonic structures in other organisms (Fristrom, 1976; Fristrom & Chihara,
1978; Keller, 1980; Poole & Steinberg, 1981; Kageyama, 1982; also see Trinkaus,
1976; 1984). While cell rearrangements in some cases appear to be passive and the
consequence of other active morphogenetic changes (Kageyama, 1982), in other
examples there is evidence that it requires the active translocation of cells past one
another (Fristrom, 1976; Fristrom & Chihara, 1978). Only in the case of imaginal
disc evagination has the question of how cells rearrange been well studied. Fristrom
(1983) has documented changes in the junctional complexes between imaginal disc
cells by freeze-fracture studies and has proposed that these changes are what are
responsible for rearrangement. Thus it is very possible that the behaviours exhibited by notochord cells as they rearrange are not related at all to their movement. Further study is needed to clarify this point for the ascidian notochord.
Subsequent to the completion of cell interdigitation, further tail elongation correlates with a shape change of the notochord cells. Videorecordings of anterior half
embryos show that notochord cells are 'pushed' out of the posterior end of the
fragment and form chains (Miyamoto, unpublished observations, see also Reverberi, 1971). The spewing out of cells occurs during the period of cell shape changes
and not during the period of cell interdigitation and suggests that they are actively
producing an elongating force. Cell rearrangement and shape changes, therefore,
appear to be principal behaviours responsible for the elongation of the ascidian tail.
Ascidians are ideal organisms to study further these behaviours and the cellular
16
DAVID M. MIYAMOTO
mechanisms that cause them. In these embryos these dynamic cell changes can be
correlated with ultrastructural changes and probed experimentally using manipulations that already have been used to study other aspects of ascidian embryogenesis.
This study was made possible in part by a Steps-Toward-Independence Fellowship from the
Marine Biological Laboratory, a University Research Council Grant from Seton Hall University,
and National Science Foundation Grant No. RII-8210021 (Equipment for Two and Four Year
Colleges and Universities) to D. M. Miyamoto. Additional support was provided by Grant No.
HD-16547 from the National Institute of Child Health and Human Development to J. R. Whittaker. We would like to thank J. R. Whittaker, Tom Meedel, and Jane Loescher of the Boston
University Marine Program for the assistance they provided. Our thanks also go to Sandy
Bochese and Pat Schall of the Seton Hall Educational Media Centre for their greatly appreciated
assistance during the preparation of the figures.
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(Accepted 2 November, 1985)