/. Embryo!. exp. Morph. Vol. 29, 2, pp. 363-382, 1973
Printed in Great Britain
363
Morphogenetic disturbances from timed inhibitions
of protein synthesis in Fundulus
By RICHARD B. CRAWFORD, 1 CHARLES E. WILDE, Jr.,
MURK H. HEINEMANN AND F. J. HENDLER
From the Department of Biology, Trinity College, Hartford, Conn.,
Department of Histology and Embryology, School of Dental Medicine
University of Pennsylvania, and the Mount Desert Island Biological
Laboratory, Salisbury Cove, Maine
SUMMARY
The reversible inhibition of protein synthesis at the 75-95 % level in the early zygote of
Fundulus results in a specific series of developmental failures dependent upon the times of
inhibitor pulse initiation. The severity of the morphogenetic failure is inversely related to the
time of initiation and directly to the length of the pulse. The defects reflect the time dependent
serial order of events in morphogenesis. The defects range from failure of cleavage through
disorders of blastulation, failure of axiation, anencephaly to microcephaly and are entirely
predictable. With the exception of cleavage failure the pattern is identical with that found
using pulses of actinomycin D in a similar manner. The agent used was pactamycin, an antibiotic which reversibly inhibits amino acid incorporation into protein by disturbing the
assembly of the functional ribosomal complex. The significance of time dependent protein
synthesis as an active expression in morphogenesis of similarly time dependent information
flow via RNA synthesis is discussed.
INTRODUCTION
Oviparous teleost fish such as Fundulus heterociitus have many advantages for
the analysis of macromolecular events in embryogenesis. Eggs are available in
large numbers. Their development can easily be made isochronous. Thus they
constitute an appropriate vertebrate system upon which molecular biological
studies can be carried out quantitatively.
In a wide variety of organisms development from fertilization to the high
blastula appears to be controlled by informational RNA previously synthesized
in the ovum and thus on maternal templates (reviewed by Davidson, 1968 and
Tyler & Tyler, 1970). The teleosts are no exception to this rule (Wilde &
Crawford, 1966, 1968; Kafiani, 1970). However, in certain teleosts an RNA
synthesis essential to normal morphogenesis begins immediately upon fertilization (Crawford & Wilde, 1966). Its expression in the control of morphogenesis
begins at gastrulation and continues from that time. The integrity of the
1
Author's address: Department of Biology, Trinity College, Hartford, Connecticut 06106,
U.S.A.
364
R. B. CRAWFORD AND OTHERS
temporal control of the serial order of morphogenetic transcription is essential
for normogenesis.
The temporal control of protein synthesis and the integrity of its serial order
in early embryogenesis may also be an essential mechanism in morphogenesis.
We report here the effects on morphogenesis of interference with protein
synthesis in the early zygote.
METHODS AND MATERIALS
Biological materials
Fundulus heteroclitus (Linnaeus) was obtained in very large numbers by seine
in estuarine waters of Frenchman Bay and kept in floating live cars in sea water.
Ripe eggs were obtained by manual stripping into membrane filtered (0-45 /im
pore size) 50 % sea water, sperm by mincing dissected testes in filtered 50 % sea
water. Gametes were mixed for 60 sec as measured by stop watch. Shortly
before the end of this period zygotes were washed quickly several times. During
the breeeding season gametes are uniformly functional. At 30 sec over 90 % of
eggs are fertilized as tested by previous experiments and by contemporary
controls. Embryos developed at 16 °C in an incubator in filtered 50 % sea water
which was changed daily.
Development was regular in controls. When care is taken, with proper culture
methods in clean water and glassware, anomalies are extremely rare. Hatching
takes place at 40 ± 2 days. Stage references are to the normal tables of Oppenheimer (1937), Armstrong & Child (1965) and the resolution of differences by
New (1966). Stage reference numbers are always referred to Oppenheimer, which
correlated with the unpublished color photographs of Wilde.
Histological preparations
Embryos were fixed in full strength formalin directly from unmanipulated
specimens. Fixation in this manner insured rapid penetration of the chorion.
Fixed specimens were then dissected free from their chorions, dehydrated in
the usual ethanol series, doubly imbedded in methyl benzoate celloidin and
paraffin and sectioned at 6/an. Sections were stained in Delafield's hematoxylin
and fast green and mounted in the usual manner.
This method of fixation was adopted since dissection in vivo of the chorion
is difficult. Prior fixation permits dissection after very delicate cellular structures
have been hardened. This tends to protect anomalous embryos from accidental
distortion and artifact production. The yolk itself still presents a problem as it
resists infiltration and has a tendency to 'shingle' or crack during sectioning.
Often such shards are deposited on top of the cellular masses, organs and parts
under study.
Protein synthesis and morphogenesis
365
Photography
Photographs of living specimens were taken using an MP-3 camera (PolaroidLand Corporation) and either Pola-color or ASA 3000 polaroid film with a
Wild-Heerbrugg dissecting microscope and a monocular tube using reflected
illumination against a black background.
Histological sections were photographed on Wratten Metallographic plates
(Eastman-Kodak) with a Zeiss Ultraphot using apochromatic objectives and an
aplanat-achromatic condenser.
Amino acid incorporation into proteins
Zygotes or embryos were washed six times with a vigorous jet of filtered 50 %
sea water, and incubated in 3 ml of 50 % sea water (incubation medium) containing 30//curies of 14C-labeled amino acids (New England Nuclear). For any
individual assay, 50-100 zygotes or embryos were used and for the bulk of the
experiments the exogenous 14C-amino acid was either uniformly labeled valine
or lysine (spec. act. 200 mCi/mmole, New England Nuclear). Incubations were
for 2 h in covered 50 ml beakers, shaking gently on a DubnofT shaker at 25 °C.
At the end of this period, the embryos were vigorously washed six times with
50 ml of 50 % filtered sea water and quickly transferred to an ice cold PotterElvehjem homogenizer to which 5-0 ml of cold 5 % trichloroacetic acid (TCA)
containing cold carrier amino acid was added. The embryos were homogenized
and the chorions separated and discarded. After standing at 0 °C for 15min
with frequent mixing, the homogenate was centrifuged and the supernatant
discarded. This procedure was continued until there was no radioactivity
(usually four washes) in the supernatant. The precipitate was resuspended in
50 ml of 5 % TCA and heated at 90 °C for 15 min. Upon cooling and centrifugation, the precipitate was further washed serially by resuspension in and
centrifugation from, 50 ml of distilled H2O, ethanol-ether (1:1 v/v) and ether.
The final pellet was dried. Solutions of approximately 1 mg/ml were made from
the protein pellet in 0-1 M-NaOH. Aliquots from these solutions were counted
in an automatic gas flow counter at 20 % efficiency. Other aliquots were
analyzed for protein concentration by the modified biuret test of Itzhaki &
Gill (1964).
Fundulus embryos are endowed with a very tough chorion which rapidly
rises from the egg at fertilization. Microbes presumably can be found attached
to its exterior but in the living zygote are prevented from contact with the blastomeres. Whatever microbiota are present external to the chorion are removed
prior to the assay by the addition of TCA and homogenization in this medium.
The chorions are split into large pieces and are removed from the homogenate
by a very mild initial centrifugation. Consequently the probability that exogenous
microbial incorporation of amino acids into protein would complicate the
results is minimal.
24
EMB 29
366
R. B. CRAWFORD AND OTHERS
Table 1. Amino acid incorporation into proteins 0/Fundulus heteroclitus
embryos*
cpm per mg protein
Stage Lysine Leucine
2
4
5
6
7
8
10
11
15
19
20
22
25
28
30
10
41
63
711
9
14
83
625
605
Valine-Lysine
Phenylalanine
750
200
20
17
2175
17
15
19
5375
3550
2014
6935
20
36
16
32
Mixture **
10
63
1865
3553
16896
Valine
15
322
227
497
517
1330
16955
8298
5011
2257
21269
18977
46130
8374
* Assays were performed as described in the text.
** A mixture of 15 amino acids (New England Nuclear) with specific activities ranging
from 118 to 410 mCi/mmole.
RESULTS
Incorporation of amino acids into Fundulus protein
A variety of exogenous amino acids are readily but differentially incorporated
into protein in normal Fundulus zygotes. In all cases the rate of incorporation
during cleavage is low but increases dramatically at the onset of gastrulation
(stage 10-11). Phenylalanine is an exception requiring further study. Pertinent
data are presented in Table 1. In the present experiments, either lysine or valine
was used as the radioactively labeled precursor supplied in the incubation
medium.
Effects of pactamycin on amino acid incorporation
Preliminary studies had indicated that pactamycin was an excellent reversible
inhibitor of protein synthesis in Fundulus embryos. In the present work, concentrations of 20/tg/ml were used in all cases since this gave maximum inhibition
and reasonable survivorship as determined previously by dose response studies
in this system. The results are recorded in Table 2. Incorporation of the labeled
precursor was inhibited at all stages. Permanent defects of morphogenesis are
brought about by this inhibitor, as will be presently described. Using both of
these criteria we conclude that the drug acts intracellularly and directly on
protein synthesis, in a manner similar to its action as reported in other systems
(for review see Cohen, Herner & Goldberg, 1969).
Protein synthesis and morphogenesis
367
Table 2. Inhibition ofvaline incorporation into proteins 0/Fundulus
embryos by pactamycin*
Developmental stage
Inhibition (%)
2 (1 cell zygote)
74
99
6-7 (16-32 cells)
96
8 (mid-blastula)
87
22 (early motile embryo)
85
24-27 (maturing fry)
77
30 (prehatch fry)
* Assays were performed as described in the text.
Table 3. Reversal of pactamycin inhibition ofvaline incorporation into
Fundulus protein*
Time removed from pactamycin (h)
cpm/mg protein
Control (no exposure to pactamycin)
8298
1097
4848
5735
11926
13111
0
1
2
4
24
* Assays were performed as described in the text. Embryos were at developmental stage 22
Reversibility of pactamycin inhibition of protein synthesis
Embryos of a wide spectrum of stages were tested and because all results
were similar we report here representative data of embryos of stage 22 (midway
in development, eyes unpigmented). These were given a 2 h pulse of pactamycin
(20//g/ml). At the end of the pulse time, the embryos were washed three times
with 50 ml of 50% sea water and 15min later this washing procedure was
repeated. At different times after removal of pactamycin the embryos were
tested for their ability to incorporate [14C]valine into protein. The results are in
Table 3. It appears that the normal rate of protein synthesis is restored within
4 h after pactamycin is removed.
Effects of pactamycin on RNA synthesis
The incorporation of [14C]uridine into Fundulus RNA was measured in the
presence of the inhibitor. Embryos of stage 26-28 were typical of a wide variety
of stages tested. The results are shown in Table 4 which also contains accompanying data for the effect of pactamycin on protein synthesis at this particular
stage. It is clear that during the experimental period, RNA synthesis in Fundulus
embryos is not significantly affected by pactamycin.
24-2
368
R. B. CRAWFORD AND OTHERS
Table 4. Effect of pactamycin on RNA synthesis in Fundulus embryos
Controls
PactamycinJ
RNA synthesis*
cpm/100 embryos
Protein synthesis!
cpm/mg protein
8482
8277
5011
537
* RNA synthesis was measured as the incorporation of 14C-labeled uridine into the hot
TCA-soluble fraction of the embryo as described in Crawford & Wilde (1966).
! Assay was performed as described in the text, using [14C]lysine as precursor.
% Pactamycin concentration was 20 /*g/ml in filtered 50 % sea water.
Table 5. Effect of pactamycin on morphogenesis of Fundulus embryos*
Refer to Fig. no.
time!
0+0
0+ i
length (min)
Developmental result
15
Moribund (1 cell) and cell mass
30
60
120
1440
15
30
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Cell mass
Cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Cell mass
Cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Barley axiate
Cell mass
Cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Barley axiate
Cell mass
Cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
Axiate-anencephalic
Cell mass
Cell mass
Moribund (1 cell) and cell mass
Moribund (1 cell)
60
0+ 1
0+2
0+3
0+4
0+5
120
1440
15
30
60
120
1440
15
30
60
120
1440
15
30
60
120
1440
15
30
60
120
1440
15
30
60
120
1440
in vivo histological
1
—
—
1
—
—
1
—
1
—
2
13
13
14, 15
2
—
—
—
14, 15
2
2
—
—
—
3
—
—
—
—
4
—
—
—
—
5
—
2
—
—
14, 15
14, 15
16, 17
—
—
—
—
18
—
•—•
—
—
19
—
14, 15
—
—
369
Protein synthesis and morphogenesis
Table 5 (cont.)
Refer to Fig. no.
Tn i ttn t i n n
XI111 lUUV/J 1
timet
0+10
0 + 30
0 + 60
Control
Pntef
X U IJV,
length (min)
15
30
60
120
1440
15
30
60
120
1440
15
30
60
120
1440
A
1
i
Developmental result
Axiate-anencephalic
Cell mass
Cell mass
Moribund (1 cell) and
Moribund (1 cell)
Normal
Normal
Axiate-anencephalic
Moribund (1 cell) and
Moribund (1 cell) and
Normal
Microcephalic
Microcephalic
Moribund (1 cell) and
Moribund (1 cell) and
Normal
cell mass
cell mass
cell mass
cell mass
cell mass
in vivo
6
1
1
—
—
8
8
9
—
—
10
11
11
—
—
12
histological
20
14, 15
14, 15
—
—
21
21
19,20
—.
—
21
—
—
—
—
21
* Fertilized eggs were placed in pactamycin solution (20//g/ml) at the indicated times
and removed to 50 % sea water after the indicated length of time.
t Initiation time is stated in reference to fertilization time, i.e. 0 + 2 indicates 2 min postfertilization.
Effects of pactamycin on morphogenesis
When zygotes or early embryos are maintained in pactamycin continuously,
mortality is 100 % within 2-4 days. However, depending upon the time after
fertilization when the inhibitor is added, certain significant observations can be
made. Generally, there is a delay in cleavage time compared to the control,
irregularity of blastomere size and irregularity of blastomere distribution over
the yolk. With an early onset of the inhibitor treatment (within 1 min of fertilization: (0+1)), no cleavage occurs although the blastodisc is raised in an
apparently normal manner. With a delay of onset of treatment varying from
(0+ 1) to (0 + 2) initial cleavages occur although they are greatly delayed. Thus
at (0+1) 15 % of the zygotes underwent the first cleavage. This was increased
to 40 % at (0+10). Delay of onset of treatment until (0 + 20) led to the accomplishment of three cleavages in 25 % of zygotes so treated.
Initiation of inhibitor treatment at any later time (i.e. beyond 0 + 20) during
cleavage permitted two or three more cleavages but the resultant 'morulae' or
'blastulae' were very abnormal. Common attributes of all were irregularity in
cell distribution and inequality in cell size.
It should be stressed that continuous incubation in pactamycin is ultimately
lethal although increase in 'free time' prior to onset of treatment tends to
permit a small number of cleavage cycles and a delay in mortality. Since the
370
R. B. CRAWFORD AND OTHERS
Protein synthesis and morphogenesis
371
pactamycin treatment at this time (stage 2) leads to 74 % inhibition of protein
synthesis, rising to 99 % at stage 6-7 (16-32 cells), we conclude that a certain
amount of immediately preceding, yet ongoing protein synthesis is required for
cleavage processes (Table 2).
Morphogenetic defects associated with variation of pactamycin pulse initiation
time and length were investigated. The pulse lengths selected were 15min,
30min, 60min, 120min and one day (1440 min). Incubations in pactamycin
were begun at 0,0 + i(i.e. 0+15 sec), 0 + 1 , 0 + 2, 0 + 3,0 + 4, 0 + 5,0+10,0 + 30
and 0 + 60 min for each pulse length. Approximately 30 embryos were used in
each case and the fluid volume in each dish was 15 ml. Daily observations were
made and survivors were fixed for histological examination at 0 + 40 days, the
onset of hatching of the controls.
The matrix of this large experiment can be followed by reference to Table 5
which will also be useful in the section on histological analysis.
During the experiment, living embryos were examined for success or failure
and degree of normality of cleavage, of formation of the high blastula, of
gastrulation and epiboly, of body axis formation and of the degree of normality
expressed in the formation of the head and head structures. These observations
were correlated with histological analyses which will be reported in the following
section.
In general the developmental failures followed the serial order and time
dependence previously reported by us for precisely timed initiations of inhibition
of RNA synthesis by actinomycin D (Wilde & Crawford, 1966). The type of
FIGURES
1-12
All figures are of living eggs and embryos. Initial magnification 50 x . Bar indicates
1 mm. All photographs taken at 30 days after fertilization.
Fig. 1. Cleavage failure due to pulse initiation 0 + 0, duration 15 min.
Fig. 2. Abnormal blastula, 'pile of cells' due to pulse initiation at 0+1, duration
15 min.
Fig. 3. Questionable axiation, pile of cells longer than wide, due to pulse initiation
at 0 + 3, duration 15 min.
Fig. 4. Barely axiate embryo due to pulse initiation at 0 + 4, duration 15 min.
Fig. 5. Axiate-anencephalic embryo due to pulse initiation at 0+5, duration 15 min.
Fig. 6. More orderly developed but still axiate-anencephalic embryo due to pulse
initiation at 0+10, duration 15 min.
Fig. 7. Non-axiate, chaotic cellular mass due to pulse initiation at 0+10, duration
30 min.
Fig. 8. Normal embryo recovered from pulse initiation time 0 + 30, duration 60 min.
Fig. 9. Axiate-anencephalic embryo due to pulse initiation at 0+30, duration 60 min.
Fig. 10. Normal embryo recovered from pulse initiation time 0+ 60, duration 15 min.
Fig. 11. Microcephalic embryo due to pulse initiation at 0 + 60, duration 30 min.
Fig. 12. Normal isochronous untreated control.
372
15
R. B. CRAWFORD AND OTHERS
Protein synthesis and morphogenesis
373
failure of morphogenesis was primarily dependent upon time of pulse initiation
with pactamycin while the severity of the defect was in part dependent upon the
length of the pulse period.
1. Pulse initiation at 0 + 0 or 0 + } led to complete failure of cleavage (Fig. 1)
in most cases, while a few showed accumulation of a 'pile' of cells.
2. Pulse initiation at 0 + 1 led to cleavage and the accumulation of a 'pile'
of cells resembling somewhat the blastula (Fig. 2). No further morphogenesis
ensued. Some embryos suffering pulse durations of 15 and 30min survived to
hatching of the controls while those exposed to pulse duration of 60, 120 and
1440 min succumbed.
3. Pulse initiation at 0 + 2 led to results similar in all observable detail to
those at 0 + 1 .
4. Pulse initiation at 0 + 3, with a duration of 15 min led to very slightly
improved morphogenesis as expressed by irregular cellular masses which were
somewhat longer than wide (Fig. 3). However, it is questionable whether this
result should be taken as evidence of body axis formation in view of the histological chaos to be described. Longer pulse durations were lethal with a gradual
mortality prior to hatching of the controls.
5. Zygotes subjected to pulse initiation at 0 + 4 gave rise to anomalous
embryos, some of which were, however, axiated, but barely so (Fig. 4). Survivors
in this class were from a pulse duration of 15 min. Longer pulses led to an increase in mortality prior to hatching of the controls.
6. Embryos subjected to pulse initiation at 0 + 5 were axiate but anencephalic,
when the pulse was of 15 min duration (Fig. 5). Longer pulse times (e.g. 60 min)
led to survivors which exhibited non-axiate cell masses similar to 0 + 1 and 0 + 2
(Fig. 2).
7. Survivors at pulse initiation time 0+10 were axiate-anencephalic embryos
FIGURES
13-17
All specimens fixed at 30 days after fertilization.
Fig. 13. Median section from zygote of pulse initiation time 0 + 0. Note failure
of cleavage and degenerating nuclear area. Initial magnification 250 x.
Fig. 14. Amorphous cell mass with no evidence of cellular differentiation or
morphogenesis from pulse initiation time of 0+1, duration 15 min. Median
section, initial magnification 100 x.
Fig. 15. Amorphous cell mass with two melanocytes (M) but no other evidence of
differentiation or morphogenesis except peripheral periblast ( ?), from pulse initiation
time of 0+1, duration 15 min. Initial magnification 250 x .
Fig. 16. Organoid development, possible notochord with sheath and beginning
vacuolization, from pulse initiation time of 0 + 3, duration 15 min. Initial magnification 400 x.
Fig. 17. Another organoid from the same specimen as Fig. 16, possibly alimentary
tract. Initial magnification 400 x .
374
R. B. CRAWFORD AND OTHERS
(Fig. 6) when duration was 15 min. Durations of 30 and 60 min led to survivors
which were non-axiate, chaotic, cellular masses (Fig. 7).
8. Pulse initiation at 0 + 30 with duration times of 15 and 30 min led to
embryos whose development was normal (Fig. 8). Pulse duration of 60 min led
to abnormal embryos of the axiate-anencephalic type (Fig. 9).
9. The final pulse initiation time studied (0 + 60), gave rise to embryos of
normal development at a pulse duration of 15 min (Fig. 10). Pulse durations
of 30 and 60 min led to the development of microcephalic embryos (Fig. 11).
The severity of microcephaly was greater with the longer pulse (Control =
Fig. 12).
It should be emphasized that all of the pulse initiation times had their onset
prior to the normal time of first cleavage (90-110 min).
The increase in severity with increase in pulse duration is compatible with the
concept that the synthesis of macromolecules important to morphogenesis is
bimodal. That is, initiation time delimits the signal type in an 'informative' and
labile mode while the increased pulse time covers and overwhelms a 'confirming'
or permanent mode. This hypothesis will be developed further in subsequent
publications.
Histological correlations
Analysis of longitudinal sections of embryos of this series corroborates and
extends the in vivo studies reported above. The serial order of time dependent
failures of morphogenesis is exemplified by Fig. 13, 0 + 0 (120 min), cleavage
failure; Figs. 14 and 15 0 + 1 (15 min) and 0 + 2 (15 min), amorphous cell mass,
gastrulation failure; Figs. 16 and 17, 0 + 3 (15 min) faulty axis formation; Fig.
18, 0 + 4 (15 min) barely axiate; Fig. 19, 0 + 5 (15 min) axiate-anencephalic
embryos; Fig. 20, 0+10 (15 min) improved axiation but retained anencephaly;
Fig. 21, 0 + 30 (15 min), 0 + 30 (30 min), 0 + 60 (15 min), control, normal
morphogenesis.
1. Cleavage failure (Figs. 1 and 13; exp. series 0 + 0, 0 + i). Examination of
serial sections of these specimens indicates that they consist of a single mass of
material with many vacuoles. There is no evidence of cleavage furrows or
membranes. Interior to the large vacuole layer the mass is more granular. These
granules are sparse until a still more central 'nuclear area' is reached. This consists of a somewhat more dense ring of larger granules. However, no nuclear
membrane can be observed. The picture is compatible with that of a dead or
dying single cell.
2. Amorphous cell mass-gastrulation failure (Figs. 2, 14 and 15; exp. series
0 + 1 and 0 + 2). Fig. 14 is a median section through a mass of cells resting as a
'high blastula' upon the yolk surface. The arrangement of cells shows no pattern
nor can any tissue structure be seen. Peripherally there are scattered cells with
greater amounts of cytoplasm and larger nuclei which are, perhaps, representative of periblast. There are also a few irregular internal spaces which, however,
Protein synthesis and morphogenesis
375
do not show cell profiles conforming to the space. There is, therefore, no
evidence of any organ or tissue differentiation although there has obviously been
successful cleavage to form the multicellular mass. Fig. 15 represents a median
section through a mass which has sunk into the yolk. The cellular pattern is
quite similar to that seen in Fig. 14. No tissue or organ patterns can be seen.
There are scattered large cells probably referable to periblast. Within the chaotic
pattern however, four melanocytes (M) have differentiated. There is no evidence
of morphogenesis. In this group (0 + 2) there is one specimen which appears to
be somewhat longer than it is wide. However, in detail it is identical to Fig. 15
in lacking any semblance of morphogenesis, tissue or organ formation. Its
dimensions are, therefore, interpreted as being the casual effects of random
growth and not of the initiation of axiation.
3. Attempts at abortive axis formation (Figs. 3, 16 and 17; exp. series 0 + 3).
Longitudinal sections near the mid-line of these specimens show certain structural
and tissue-like differentiations within the prevailing histological chaos. The
first to appear is an irregular cellular rod, the cells of which are often vacuolated
in a manner reminiscent of early notochordal cells (Fig. 16). The rod is quite
irregular with constrictions, bends and twists. It is bounded by a well defined
'membrane' again reminiscent of notochord sheath. It lies in the long axis of
the cellular mass. Nearby a second organoid structure is present (Fig. 17).
This represents a rather regular lumen surrounded by somewhat columnar cells
of an epithelial appearance. By comparison with control specimens, this structure appears to represent poorly differentiated alimentary tract. There is no
evidence of somites or nervous tissue. Aside from occasional pigment cells, no
other identifiable cell types or tissues are to be seen.
4. Embryos definitively but irregularly and barely axiate (Figs. 4 and 18; exp.
series 0 + 4). Sections from embryos of this series show definite axial structures
but they remain irregular and their pattern is obscure. The following tissues can
be definitely identified. Notochord (N) and sheath with vacuolated cells, alimentary tract with columnar, epithelial cells, well matured and polarized, blood
vessels and erythrocytes. There are many other organoid structures of a glandular nature and perhaps some very poorly organized nervous tissue. In
longitudinal sections one still cannot identify anterior and posterior. No somites
or muscle blocks are to be found. Pigment cells are numerous. The cellular
material between the lobes and identifiable tissues remains irregular and randomly disposed.
5. Embryos axiate but anencephalic (Figs. 5 and 19; exp. series 0 + 5). In
examining longitudinal sections such as shown in Fig. 19 it is tempting to identify
the 'head' of the embryo at the bottom and the 'tail' at the top. Caution is
required however. Such embryos have a well developed notochord and sheath
(A0, an alimentary tract (^4) with columnar epithelium and blood vessels containing erythrocytes. Nervous tissue is not easily identifiable but, perhaps, is
represented by dense aggregations such as NV. Branchial arches cannot be
376
19
R. B. CRAWFORD AND OTHERS
20
Protein synthesis and morphogenesis
311
made out nor can regularly arranged somitic muscles. Notochord is present
throughout the bulk of the section and is more highly differentiated (vacuolated)
toward the top. Very tentatively we identify the top of the figure as anterior.
There is no brain and no skull. The embryo is completely anencephalic.
6. Embryos axiate with improved organogenesis but relatively anencephalic
(Figs. 6 and 20; exp. series 0+10). The longitudinal section represented by
Fig. 20 illustrates a much less abnormal embryo. A well differentiated notochord
(N) is present which in other sections can be demonstrated to run from the large
vesicle (V) throughout the embryo. The vesicle area with poorly differentiated
nervous tissue to the left {NV) and a similar mass to the right constitutes the
anterior end. No skeletal elements of the skull are present. There is a well
differentiated alimentary tract (A) with polarized columnar epithelium. The
ventral diverticulum of glandular cells (L) may represent the liver while the dorsal
small glandular mass (F) may represent pancreas. Posteriorly and dorsally,
poorly formed muscles of somitic origin (S) are present. The vesicle (V) is
abnormal and cannot be identified. Posteriorly, just above the notochord,
ependyma of the ventral aspect of the spinal cord appears (E). There are
numerous pigment cells and fragile blood vessels of various sizes containing
erythrocytes. In the 'head' region are numerous clusters of large cells with
abundant cytoplasm and large nuclei which resemble periblast. If they indeed
represent periblast, such invasion of the embryo is a surprising phenomenon.
However, further studies on abnormal periblast are required.
7. Embryos with normal morphogenesis (Figs. 8, 10, 12 and 21; exp. series
0 + 30, 0 + 60 and controls). Fig. 21 represents a longitudinal section of a control
embryo fixed just prior to hatching and is representative of specimens of 0 + 30
(15min), 0 + 30 (30min) and 0 + 60 (15min). The organs and tissues which
result from conditions of normal morphogenesis may be usefully compared with
the previous figures in assessing the sedation of developmental defects in the
previously described experimental series.
By reference to Table 5 it will be recalled that increase in pulse duration of
FIGURES
18-20
All specimens fixed at 30 days after fertilization.
Fig. 18. Median (longitudinal?) section of possibly axiate embryo showing notochord (N) with sheath and irregular course, and possible alimentary tract (A) from
pulse initiation at 0+4, duration 15 min. Initial magnification 100 x.
Fig. 19. Longitudinal section of barely axiate embryo. Anterior end is probably at
top of picture. Poorly formed organs are seen: (NV) nervous tissue without apparent
organization; (N) notochord; (A) alimentary tract. Initial magnification 100 x.
Fig. 20. Longitudinal section of axiate anencephalic embryo. Anterior pole at top
offigure.Cephalic region is undeveloped. (NV) amorphous nerve tissue; (^unidentifiable vesicle; (P) pancreatic Anlage (?); (L) liver Anlage (?); (E) ependyma;
(/V) notochord; (A) alimentary tract; (5) poorly formed somites. Initial magnification
100 x .
378
R. B. C R A W F O R D AND OTHERS
Fig. 21. Longitudinal section of isochronous control embryo (30 days). Initial
magnification 100 x.
pactamycin tends to shift the resultant anomalous embryos toward the beginning of the serial order of defects with a similarity to earlier pulse initiation
time. Thus, 0 + 5 (60 min) is like the 0 + 2 series; 0 +10 (30 min) and 0+10 (60
min) are similar to the 0 + 2 series. The class 0 + 30 (60 min) is similar to the
0+10 series, while 0 + 60 (30 min) and 0 + 60 (60 min) are microcephalic rather
than anencephalic.
Protein synthesis and morphogenesis
379
DISCUSSION
Pactamycin has previously been demonstrated to be an inhibitor of protein
synthesis in mammalian (Colombo, Felicetti & Baglioni, 1966 and Felicetti,
Colombo & Baglioni, 1966) and bacterial (Cohen, Herner & Goldberg, 1969)
systems. The site of action has been shown to be at the binding of aminoacyltransfer RNA to ribosomal subunits (Cohen, Goldberg & Herner, 1969). The
inhibitor has now been shown to be a useful and effective agent for studies of
the control of protein synthesis in embryos of Fundulus heteroclitus. It inhibits
protein synthesis at all stages of development at the level of 75 % or greater as
expressed by a decrease in incorporation of radioactive amino acids into trichloroacetic acid-insoluble protein. Furthermore, its effect on protein synthesis is
reversible, which allows studies of the effect of inhibitions at particular periods
of synthesis in relation to morphogenesis. Since a pulse period of two hours or
less of pactamycin does not effect RNA synthesis, a clear distinction between
effects referable to translation rather than transcription can be made.
Failures of morphogenesis due to precisely timed pulses of pactamycin fall in
a serial order whose regular sequence is predictable and closely related to a
similar serial order elucidated by inhibition of RNA synthesis with actinomycin D.
It is this predictable sequence of morphogenetic failures due to precisely timed
additions of pactamycin to the incubation medium of the embryos that we wish
to stress at this time in correlation with our previous data on the morphogenetic
defects related to RNA synthesis inhibition (Wilde & Crawford, 1966, 1968).
Development to the high blastula (stage 9-10) in Fundulus is independent of
contemporary RNA synthesis on zygotic templates although in the zygote there
is initiated synthesis of RNA meaningful for the post-blastula period beginning
within minutes following fertilization. The data for Fundulus are consistent with
the findings in a broad range of embryonic systems (Davidson, 1968). The
conclusion generally held is that genomic transcription of informational molecules required for cleavage occurs prior to fertilization and thus on maternal
templates. This would include active templates for proteins concerned with
cleavage spindles, chromosome replication and increment of cell surface. This
has recently been established in echinoderms (Raff, Colot, Selvig & Gross,
1972).
Inhibition of protein synthesis through administration of pactamycin immediately upon fertilization aborts the first cleavage. If the pulse initiation is
delayed an effect decreasing in intensity is observed up to a pulse initiation time
of 20 min following fertilization. In the latter circumstances, sparing the first
10 to 20 min permits some zygotes to undergo two or three cleavages. Therefore
during this time anticipatory syntheses of cleavage associated proteins are taking
place presumably upon maternal templates. Anticipatory syntheses appear to
be required for cleavages two or three cycles in the future. Therefore, an ongoing
protein synthesis is required in Fundulus for normal development in the cleavage
380
R. B. CRAWFORD AND OTHERS
Table 6. Comparison of actinomycin D andpactamycin effects
on Fundulus morphogenesis*
Initiation
timej
0-1
1-2
2-3
3-4
4-5
5-10
10-30
30-60
Effect on morphogenesis
Inhibitor
A
-———
,
Actinomycin D Pactamycin
Prevention of early cleavage
Defective blastula
Defective gastrulation
Defective axiation
Anencephaly
Less severe anencephaly
Microcephaly
Normal development
* Compilation of observations from this paper and Wilde & Crawford (1966).
t Initiation time refers to the minutes post-fertilization when incubation in the inhibitor
began.
period. Actinomycin D has no such effect. In the presence of this drug, cleavage
to the high blastula is normal in form and in timing.
We have previously reported (Wilde & Crawford, 1966) a serial order of
defects in morphogenesis of high predictability dependent strictly upon the time
of initiation of actinomycin D inhibition of RNA synthesis. The data presented
in this paper demonstrate the similarity of morphogenetic defects beyond the
high blastula conferred by pulses of pactamycin and actinomycin D. These
correlations are shown in Table 6. The morphogenetic defects are expressed
morphologically 40 or more hours after the cessation of the inhibitory pulse
which led to the defect. While protein synthesis as a general phenomenon is
much more rapidly restored following pulse termination, the morphogenetic
defects are permanent.
We are thus drawn to the conclusion that specific protein syntheses, required
for normal morphogenesis beyond the blastula, are initiated prior to the second
minute following fertilization and thus upon zygotic templates (or alternatively
on certain maternal templates stimulated to function by the act of fertilization).
Furthermore, in dealing with the minimal pulse times in these experiments
(15min), the effect on any particular synthesis must be complete by the termination of the pulse or shortly thereafter since protein synthesis as measured
by standard methods will be resumed.
Protein synthesis is low during early zygotic periods as reflected in the data.
Much of this synthesis must necessarily be concerned with spindle protein and
histone synthesis, to name the most obvious. It would appear therefore that the
concurrent protein synthesis required for post-blastula morphogenesis takes
place at a very low level. If informational RNA is presumed to be attached to
appropriate ribosomal configurations and the presence of potentially functional
Protein synthesis and morphogenesis
381
ribosomal units is assumed, upon relief of the inhibition, why is subsequent
morphogenesis disturbed? Perhaps pactamycin firmly enters the functional unit
and renders it inactive on a relatively permanent basis. Consequently, since a
serial order of RNA synthesis essential to post-blastula morphogenesis has been
demonstrated to be time dependent, the essential first morphogenetically meaningful proteins are never given the morphogenetically correct, temporal opportunity to be synthesized, their templates having been passed by in time and,
perhaps, degraded. Relief of the inhibition may allow for all the essential protein
syntheses, certain of which are now out of proper sequence and relationship to
the ongoing development. Under these conditions they cannot play their normal
morphogenetic role.
It follows, in the major axial systems studied here in Fundulus, that early
syntheses are successionally dependent upon antecedent ones. Only after the
fifth minute is there any expression of reasonably normal morphogenesis of one
part (viz. gut) while another part (the brain) remains utterly defective. Morphogenetically meaningful macromolecular syntheses appear to fall into a ramifying
scheme which is initially one tract prior to branching. Organogenesis of a
particular structure would be inhibited by failure of antecedent syntheses at a
branch point. Under such circumstances, after the fifth minute, inhibition along
one ramus would lead to the failure of morphogenesis of subsequent dependent
development while along an uninhibited branch morphogenesis would continue
more or less normally. Such a scheme however, must be broad enough conceptually to include connecting links at varying levels of organogenesis.
These data are consistent with the conclusion that morphogenesis in Fundulus
is under genomic control and that the control functions through the well
established mechanisms of transcription and translation. They further indicate
that the macromolecular chemistry of morphogenesis begins within seconds of
fertilization at least as expressed in the primary and major morphogenetic
activities of the zygote. The expression of genomic control as reflected in the
time dependent serial order of developmental failures here analyzed indicates
that morphogenetic chemistry proceeds rapidly and stepwise. Failure at early
steps causes abnormal orientation of any further development.
Yet it is of intense interest that development does in fact continue, carrying
the immutable defects, notochord without nervous system, nervous system
without somites, etc. The morphogenetic program entered into at fertilization
behaves as though all cells and their progeny were committed and cognizant
of time flow and position; only those cells which were the open targets of the
inhibitor during, and only during, the pulse failed in morphogenetic commitment and behaviour.
It has long been considered that morphogenesis is initiated at gastrulation via
inductive processes. We wish to emphasize that the classic phenomenology is,
in Fundulus, preceded by 40 h of essential macromolecular synthesis without
which morphogenesis is aborted. Indeed the most important initiating and
25
EM B 29
382
R. B. CRAWFORD AND OTHERS
controlling events appear to be confined to the period immediately following
fertilization and well within the first cleavage period while the zygote is a single
cell.
The specific protein syntheses characteristic of cellular differentiation are
expressed in many of the terata discussed here. These are also presumably under
genomic control. However, the transcription and translation processes for cellular
differentiation appear to be initiated later in embryogenesis beyond the period
of morphogenetic determination. The relationship of primary morphogenetic
processes to the chemistry of cellular differentiation remains to be explored.
The authors wish to thank Mrs Diane Zucker and Mrs Michele Koppelman for their
excellent technical assistance. Gift of pactamycin from the Upjohn Co. was very much
appreciated.
The work reported here was supported by N.S.F. Grant GB-6766 and an NSF Grant to
the Mount Desert Island Biological Laboratory GB-28139.
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{Received 18 May 1972, revised 30 August 1972)