morphogenetic disturbances from timed inhibitions of ...fundulus heteroclitus (linnaeus) was...

/ . Embryo!. exp. Morph. Vol. 29, 2, pp. 363-382, 1973 3 6 3

Printed in Great Britain

Morphogenetic disturbances from timed inhibitionsof 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 BiologicalLaboratory, Salisbury Cove, Maine

SUMMARY

The reversible inhibition of protein synthesis at the 75-95 % level in the early zygote ofFundulus results in a specific series of developmental failures dependent upon the times ofinhibitor pulse initiation. The severity of the morphogenetic failure is inversely related to thetime of initiation and directly to the length of the pulse. The defects reflect the time dependentserial order of events in morphogenesis. The defects range from failure of cleavage throughdisorders of blastulation, failure of axiation, anencephaly to microcephaly and are entirelypredictable. With the exception of cleavage failure the pattern is identical with that foundusing pulses of actinomycin D in a similar manner. The agent used was pactamycin, an anti-biotic which reversibly inhibits amino acid incorporation into protein by disturbing theassembly of the functional ribosomal complex. The significance of time dependent proteinsynthesis as an active expression in morphogenesis of similarly time dependent informationflow via RNA synthesis is discussed.

INTRODUCTION

Oviparous teleost fish such as Fundulus heterociitus have many advantages forthe analysis of macromolecular events in embryogenesis. Eggs are available inlarge numbers. Their development can easily be made isochronous. Thus theyconstitute an appropriate vertebrate system upon which molecular biologicalstudies can be carried out quantitatively.

In a wide variety of organisms development from fertilization to the highblastula appears to be controlled by informational RNA previously synthesizedin the ovum and thus on maternal templates (reviewed by Davidson, 1968 andTyler & Tyler, 1970). The teleosts are no exception to this rule (Wilde &Crawford, 1966, 1968; Kafiani, 1970). However, in certain teleosts an RNAsynthesis essential to normal morphogenesis begins immediately upon fertili-zation (Crawford & Wilde, 1966). Its expression in the control of morphogenesisbegins 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 essentialfor normogenesis.

The temporal control of protein synthesis and the integrity of its serial orderin early embryogenesis may also be an essential mechanism in morphogenesis.We report here the effects on morphogenesis of interference with proteinsynthesis in the early zygote.

METHODS AND MATERIALS

Biological materials

Fundulus heteroclitus (Linnaeus) was obtained in very large numbers by seinein 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 /impore size) 50 % sea water, sperm by mincing dissected testes in filtered 50 % seawater. Gametes were mixed for 60 sec as measured by stop watch. Shortlybefore the end of this period zygotes were washed quickly several times. Duringthe breeeding season gametes are uniformly functional. At 30 sec over 90 % ofeggs are fertilized as tested by previous experiments and by contemporarycontrols. Embryos developed at 16 °C in an incubator in filtered 50 % sea waterwhich was changed daily.

Development was regular in controls. When care is taken, with proper culturemethods in clean water and glassware, anomalies are extremely rare. Hatchingtakes place at 40 ± 2 days. Stage references are to the normal tables of Oppen-heimer (1937), Armstrong & Child (1965) and the resolution of differences byNew (1966). Stage reference numbers are always referred to Oppenheimer, whichcorrelated with the unpublished color photographs of Wilde.

Histological preparations

Embryos were fixed in full strength formalin directly from unmanipulatedspecimens. Fixation in this manner insured rapid penetration of the chorion.Fixed specimens were then dissected free from their chorions, dehydrated inthe usual ethanol series, doubly imbedded in methyl benzoate celloidin andparaffin and sectioned at 6/an. Sections were stained in Delafield's hematoxylinand fast green and mounted in the usual manner.

This method of fixation was adopted since dissection in vivo of the chorionis difficult. Prior fixation permits dissection after very delicate cellular structureshave been hardened. This tends to protect anomalous embryos from accidentaldistortion and artifact production. The yolk itself still presents a problem as itresists infiltration and has a tendency to 'shingle' or crack during sectioning.Often such shards are deposited on top of the cellular masses, organs and partsunder study.

Protein synthesis and morphogenesis 365

Photography

Photographs of living specimens were taken using an MP-3 camera (Polaroid-Land Corporation) and either Pola-color or ASA 3000 polaroid film with aWild-Heerbrugg dissecting microscope and a monocular tube using reflectedillumination against a black background.

Histological sections were photographed on Wratten Metallographic plates(Eastman-Kodak) with a Zeiss Ultraphot using apochromatic objectives and anaplanat-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) con-taining 30//curies of 14C-labeled amino acids (New England Nuclear). For anyindividual assay, 50-100 zygotes or embryos were used and for the bulk of theexperiments the exogenous 14C-amino acid was either uniformly labeled valineor lysine (spec. act. 200 mCi/mmole, New England Nuclear). Incubations werefor 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 with50 ml of 50 % filtered sea water and quickly transferred to an ice cold Potter-Elvehjem homogenizer to which 5-0 ml of cold 5 % trichloroacetic acid (TCA)containing cold carrier amino acid was added. The embryos were homogenizedand the chorions separated and discarded. After standing at 0 °C for 15minwith frequent mixing, the homogenate was centrifuged and the supernatantdiscarded. This procedure was continued until there was no radioactivity(usually four washes) in the supernatant. The precipitate was resuspended in50 ml of 5 % TCA and heated at 90 °C for 15 min. Upon cooling and centri-fugation, the precipitate was further washed serially by resuspension in andcentrifugation 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 fromthe protein pellet in 0-1 M-NaOH. Aliquots from these solutions were countedin an automatic gas flow counter at 20 % efficiency. Other aliquots wereanalyzed for protein concentration by the modified biuret test of Itzhaki &Gill (1964).

Fundulus embryos are endowed with a very tough chorion which rapidlyrises from the egg at fertilization. Microbes presumably can be found attachedto its exterior but in the living zygote are prevented from contact with the blasto-meres. Whatever microbiota are present external to the chorion are removedprior 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 homogenateby a very mild initial centrifugation. Consequently the probability that exogenousmicrobial incorporation of amino acids into protein would complicate theresults is minimal.

24 EMB 29


Table 1. Amino acid incorporation into proteins 0/Fundulus heteroclitusembryos*

Stage

245678

101115192022252830

Lysine

104163

711

625

2014

6935

50112257

Leucine

91483

605

21269

cpm per

Valine-Lysine

750200

2175

53753550

18977

mg protein

Phenylalanine

2017

171519

20361632

Mixture **

1063

1865

355316896

16955

46130

Valine

15

322227497517

1330

8298

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 incorporatedinto protein in normal Fundulus zygotes. In all cases the rate of incorporationduring cleavage is low but increases dramatically at the onset of gastrulation(stage 10-11). Phenylalanine is an exception requiring further study. Pertinentdata are presented in Table 1. In the present experiments, either lysine or valinewas used as the radioactively labeled precursor supplied in the incubationmedium.

Effects of pactamycin on amino acid incorporation

Preliminary studies had indicated that pactamycin was an excellent reversibleinhibitor of protein synthesis in Fundulus embryos. In the present work, con-centrations of 20/tg/ml were used in all cases since this gave maximum inhibitionand reasonable survivorship as determined previously by dose response studiesin this system. The results are recorded in Table 2. Incorporation of the labeledprecursor was inhibited at all stages. Permanent defects of morphogenesis arebrought about by this inhibitor, as will be presently described. Using both ofthese criteria we conclude that the drug acts intracellularly and directly onprotein synthesis, in a manner similar to its action as reported in other systems(for review see Cohen, Herner & Goldberg, 1969).


Table 2. Inhibition ofvaline incorporation into proteins 0/Fundulusembryos by pactamycin*

Developmental stage

2 (1 cell zygote)6-7 (16-32 cells)8 (mid-blastula)22 (early motile embryo)24-27 (maturing fry)30 (prehatch fry)

Inhibition (%)

749996878577

* Assays were performed as described in the text.

Table 3. Reversal of pactamycin inhibition ofvaline incorporation intoFundulus protein*

Time removed from pactamycin (h) cpm/mg protein

Control (no exposure to pactamycin)0124

24

8298109748485735

1192613111

* 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 resultswere similar we report here representative data of embryos of stage 22 (midwayin 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 timeswith 50 ml of 50% sea water and 15min later this washing procedure wasrepeated. At different times after removal of pactamycin the embryos weretested for their ability to incorporate [14C]valine into protein. The results are inTable 3. It appears that the normal rate of protein synthesis is restored within4 h after pactamycin is removed.

Effects of pactamycin on RNA synthesis

The incorporation of [14C]uridine into Fundulus RNA was measured in thepresence of the inhibitor. Embryos of stage 26-28 were typical of a wide varietyof stages tested. The results are shown in Table 4 which also contains accompany-ing data for the effect of pactamycin on protein synthesis at this particularstage. It is clear that during the experimental period, RNA synthesis in Fundulusembryos is not significantly affected by pactamycin.

24-2


Table 4. Effect of pactamycin on RNA synthesis in Fundulus embryos

RNA synthesis* Protein synthesis!cpm/100 embryos cpm/mg protein

Controls 8482 5011PactamycinJ 8277 537

* RNA synthesis was measured as the incorporation of 14C-labeled uridine into the hotTCA-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! length (min) Developmental result in vivo histological

0 + 0

0 + i

0 + 1

0 + 2

0 + 3

0+4

0 + 5

153060

1201440

153060

1201440

153060

1201440

153060

1201440

153060

1201440

153060

1201440

153060

1201440

Moribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell)Moribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell)Cell massCell massMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell)Cell massCell massMoribund (1 cell) and cellMoribund (1 cell) and cellMoribund (1 cell)Barley axiateCell massCell massMoribund (1 cell) and cellMoribund (1 cell)Barley axiateCell massCell massMoribund (1 cell) and cellMoribund (1 cell)Axiate-anencephalicCell massCell massMoribund (1 cell) and cellMoribund (1 cell)

massmassmassmass

massmassmassmass

massmass

massmass

mass

mass

mass

1——

1——

1—

1—22

———

22

———3

————4

————

5—2

——

13

13

14,14,

14,14,

16,————

18—•—•——19—

14,——

1515

1515

17

15


Tn i ttn t i n nXI111 lUUV/J 1

timet

0+10

0 + 30

0 + 60

Control

PntefX U IJV,

length

153060

1201440

153060

1201440

153060

1201440

Table 5 (cont.)

(min) Developmental result

Axiate-anencephalicCell massCell massMoribund (1 cell) and cell massMoribund (1 cell)

NormalNormalAxiate-anencephalicMoribund (1 cell) and cell massMoribund (1 cell) and cell mass

NormalMicrocephalicMicrocephalicMoribund (1 cell) and cell massMoribund (1 cell) and cell mass

Normal

Refer

i

in vivo

611

——

889

——

101111—

—

12

to Fig. no.A

1

histological

2014, 1514, 15——

212119,20—.—

21————

21

* Fertilized eggs were placed in pactamycin solution (20//g/ml) at the indicated timesand 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 post-fertilization.

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 afterfertilization when the inhibitor is added, certain significant observations can bemade. Generally, there is a delay in cleavage time compared to the control,irregularity of blastomere size and irregularity of blastomere distribution overthe yolk. With an early onset of the inhibitor treatment (within 1 min of ferti-lization: (0+1)), no cleavage occurs although the blastodisc is raised in anapparently normal manner. With a delay of onset of treatment varying from(0+ 1) to (0 + 2) initial cleavages occur although they are greatly delayed. Thusat (0+1) 15 % of the zygotes underwent the first cleavage. This was increasedto 40 % at (0+10). Delay of onset of treatment until (0 + 20) led to the accom-plishment of three cleavages in 25 % of zygotes so treated.

Initiation of inhibitor treatment at any later time (i.e. beyond 0 + 20) duringcleavage permitted two or three more cleavages but the resultant 'morulae' or'blastulae' were very abnormal. Common attributes of all were irregularity incell distribution and inequality in cell size.

It should be stressed that continuous incubation in pactamycin is ultimatelylethal although increase in 'free time' prior to onset of treatment tends topermit a small number of cleavage cycles and a delay in mortality. Since the


pactamycin treatment at this time (stage 2) leads to 74 % inhibition of proteinsynthesis, rising to 99 % at stage 6-7 (16-32 cells), we conclude that a certainamount of immediately preceding, yet ongoing protein synthesis is required forcleavage processes (Table 2).

Morphogenetic defects associated with variation of pactamycin pulse initiationtime and length were investigated. The pulse lengths selected were 15min,30min, 60min, 120min and one day (1440 min). Incubations in pactamycinwere begun at 0,0 + i(i.e. 0+15 sec), 0 + 1 , 0 + 2, 0 + 3,0 + 4, 0 + 5,0+10,0 + 30and 0 + 60 min for each pulse length. Approximately 30 embryos were used ineach case and the fluid volume in each dish was 15 ml. Daily observations weremade and survivors were fixed for histological examination at 0 + 40 days, theonset of hatching of the controls.

The matrix of this large experiment can be followed by reference to Table 5which will also be useful in the section on histological analysis.

During the experiment, living embryos were examined for success or failureand degree of normality of cleavage, of formation of the high blastula, ofgastrulation and epiboly, of body axis formation and of the degree of normalityexpressed in the formation of the head and head structures. These observationswere correlated with histological analyses which will be reported in the followingsection.

In general the developmental failures followed the serial order and timedependence previously reported by us for precisely timed initiations of inhibitionof 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 indicates1 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, duration15 min.Fig. 3. Questionable axiation, pile of cells longer than wide, due to pulse initiationat 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 pulseinitiation at 0+10, duration 15 min.Fig. 7. Non-axiate, chaotic cellular mass due to pulse initiation at 0+10, duration30 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.


15


failure of morphogenesis was primarily dependent upon time of pulse initiationwith pactamycin while the severity of the defect was in part dependent upon thelength 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 morphogenesisensued. Some embryos suffering pulse durations of 15 and 30min survived tohatching of the controls while those exposed to pulse duration of 60, 120 and1440 min succumbed.

3. Pulse initiation at 0 + 2 led to results similar in all observable detail tothose at 0 + 1 .

4. Pulse initiation at 0 + 3, with a duration of 15 min led to very slightlyimproved morphogenesis as expressed by irregular cellular masses which weresomewhat longer than wide (Fig. 3). However, it is questionable whether thisresult should be taken as evidence of body axis formation in view of the histo-logical chaos to be described. Longer pulse durations were lethal with a gradualmortality prior to hatching of the controls.

5. Zygotes subjected to pulse initiation at 0 + 4 gave rise to anomalousembryos, some of which were, however, axiated, but barely so (Fig. 4). Survivorsin this class were from a pulse duration of 15 min. Longer pulses led to an in-crease 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 failureof cleavage and degenerating nuclear area. Initial magnification 250 x.Fig. 14. Amorphous cell mass with no evidence of cellular differentiation ormorphogenesis from pulse initiation time of 0+1, duration 15 min. Mediansection, initial magnification 100 x.Fig. 15. Amorphous cell mass with two melanocytes (M) but no other evidence ofdifferentiation or morphogenesis except peripheral periblast ( ?), from pulse initiationtime of 0+1, duration 15 min. Initial magnification 250 x .Fig. 16. Organoid development, possible notochord with sheath and beginningvacuolization, from pulse initiation time of 0 + 3, duration 15 min. Initial magnifi-cation 400 x.Fig. 17. Another organoid from the same specimen as Fig. 16, possibly alimentarytract. Initial magnification 400 x .


(Fig. 6) when duration was 15 min. Durations of 30 and 60 min led to survivorswhich were non-axiate, chaotic, cellular masses (Fig. 7).

8. Pulse initiation at 0 + 30 with duration times of 15 and 30 min led toembryos whose development was normal (Fig. 8). Pulse duration of 60 min ledto abnormal embryos of the axiate-anencephalic type (Fig. 9).

9. The final pulse initiation time studied (0 + 60), gave rise to embryos ofnormal development at a pulse duration of 15 min (Fig. 10). Pulse durationsof 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 onsetprior to the normal time of first cleavage (90-110 min).

The increase in severity with increase in pulse duration is compatible with theconcept that the synthesis of macromolecules important to morphogenesis isbimodal. That is, initiation time delimits the signal type in an 'informative' andlabile mode while the increased pulse time covers and overwhelms a 'confirming'or permanent mode. This hypothesis will be developed further in subsequentpublications.

Histological correlations

Analysis of longitudinal sections of embryos of this series corroborates andextends the in vivo studies reported above. The serial order of time dependentfailures of morphogenesis is exemplified by Fig. 13, 0 + 0 (120 min), cleavagefailure; 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-anencephalicembryos; 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, normalmorphogenesis.

1. Cleavage failure (Figs. 1 and 13; exp. series 0 + 0, 0 + i). Examination ofserial sections of these specimens indicates that they consist of a single mass ofmaterial with many vacuoles. There is no evidence of cleavage furrows ormembranes. Interior to the large vacuole layer the mass is more granular. Thesegranules are sparse until a still more central 'nuclear area' is reached. This con-sists of a somewhat more dense ring of larger granules. However, no nuclearmembrane can be observed. The picture is compatible with that of a dead ordying single cell.

2. Amorphous cell mass-gastrulation failure (Figs. 2, 14 and 15; exp. series0 + 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 patternnor can any tissue structure be seen. Peripherally there are scattered cells withgreater amounts of cytoplasm and larger nuclei which are, perhaps, representa-tive of periblast. There are also a few irregular internal spaces which, however,


do not show cell profiles conforming to the space. There is, therefore, noevidence of any organ or tissue differentiation although there has obviously beensuccessful cleavage to form the multicellular mass. Fig. 15 represents a mediansection through a mass which has sunk into the yolk. The cellular pattern isquite 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 chaoticpattern however, four melanocytes (M) have differentiated. There is no evidenceof morphogenesis. In this group (0 + 2) there is one specimen which appears tobe somewhat longer than it is wide. However, in detail it is identical to Fig. 15in lacking any semblance of morphogenesis, tissue or organ formation. Itsdimensions are, therefore, interpreted as being the casual effects of randomgrowth 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 structuraland tissue-like differentiations within the prevailing histological chaos. Thefirst to appear is an irregular cellular rod, the cells of which are often vacuolatedin a manner reminiscent of early notochordal cells (Fig. 16). The rod is quiteirregular with constrictions, bends and twists. It is bounded by a well defined'membrane' again reminiscent of notochord sheath. It lies in the long axis ofthe cellular mass. Nearby a second organoid structure is present (Fig. 17).This represents a rather regular lumen surrounded by somewhat columnar cellsof an epithelial appearance. By comparison with control specimens, this struc-ture appears to represent poorly differentiated alimentary tract. There is noevidence of somites or nervous tissue. Aside from occasional pigment cells, noother 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 structuresbut they remain irregular and their pattern is obscure. The following tissues canbe definitely identified. Notochord (N) and sheath with vacuolated cells, alimen-tary tract with columnar, epithelial cells, well matured and polarized, bloodvessels and erythrocytes. There are many other organoid structures of a glan-dular nature and perhaps some very poorly organized nervous tissue. Inlongitudinal sections one still cannot identify anterior and posterior. No somitesor muscle blocks are to be found. Pigment cells are numerous. The cellularmaterial between the lobes and identifiable tissues remains irregular and ran-domly disposed.

5. Embryos axiate but anencephalic (Figs. 5 and 19; exp. series 0 + 5). Inexamining longitudinal sections such as shown in Fig. 19 it is tempting to identifythe 'head' of the embryo at the bottom and the 'tail' at the top. Caution isrequired however. Such embryos have a well developed notochord and sheath(A0, an alimentary tract (̂ 4) with columnar epithelium and blood vessels con-taining erythrocytes. Nervous tissue is not easily identifiable but, perhaps, isrepresented by dense aggregations such as NV. Branchial arches cannot be


19 20


made out nor can regularly arranged somitic muscles. Notochord is presentthroughout 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 byFig. 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 largevesicle (V) throughout the embryo. The vesicle area with poorly differentiatednervous tissue to the left {NV) and a similar mass to the right constitutes theanterior end. No skeletal elements of the skull are present. There is a welldifferentiated alimentary tract (A) with polarized columnar epithelium. Theventral diverticulum of glandular cells (L) may represent the liver while the dorsalsmall glandular mass (F) may represent pancreas. Posteriorly and dorsally,poorly formed muscles of somitic origin (S) are present. The vesicle (V) isabnormal and cannot be identified. Posteriorly, just above the notochord,ependyma of the ventral aspect of the spinal cord appears (E). There arenumerous pigment cells and fragile blood vessels of various sizes containingerythrocytes. In the 'head' region are numerous clusters of large cells withabundant cytoplasm and large nuclei which resemble periblast. If they indeedrepresent 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. series0 + 30, 0 + 60 and controls). Fig. 21 represents a longitudinal section of a controlembryo 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 whichresult from conditions of normal morphogenesis may be usefully compared withthe previous figures in assessing the sedation of developmental defects in thepreviously 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 noto-chord (N) with sheath and irregular course, and possible alimentary tract (A) frompulse initiation at 0+4, duration 15 min. Initial magnification 100 x.Fig. 19. Longitudinal section of barely axiate embryo. Anterior end is probably attop of picture. Poorly formed organs are seen: (NV) nervous tissue without apparentorganization; (N) notochord; (A) alimentary tract. Initial magnification 100 x.Fig. 20. Longitudinal section of axiate anencephalic embryo. Anterior pole at topof figure. Cephalic region is undeveloped. (NV) amorphous nerve tissue; (^uniden-tifiable vesicle; (P) pancreatic Anlage (?); (L) liver Anlage (?); (E) ependyma;(/V) notochord; (A) alimentary tract; (5) poorly formed somites. Initial magnification100 x.


Fig. 21. Longitudinal section of isochronous control embryo (30 days). Initialmagnification 100 x.

pactamycin tends to shift the resultant anomalous embryos toward the begin-ning of the serial order of defects with a similarity to earlier pulse initiationtime. Thus, 0 + 5 (60 min) is like the 0 + 2 series; 0 +10 (30 min) and 0+10 (60min) are similar to the 0 + 2 series. The class 0 + 30 (60 min) is similar to the0+10 series, while 0 + 60 (30 min) and 0 + 60 (60 min) are microcephalic ratherthan anencephalic.


DISCUSSION

Pactamycin has previously been demonstrated to be an inhibitor of proteinsynthesis 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 aminoacyl-transfer RNA to ribosomal subunits (Cohen, Goldberg & Herner, 1969). Theinhibitor has now been shown to be a useful and effective agent for studies ofthe control of protein synthesis in embryos of Fundulus heteroclitus. It inhibitsprotein synthesis at all stages of development at the level of 75 % or greater asexpressed by a decrease in incorporation of radioactive amino acids into trich-loroacetic acid-insoluble protein. Furthermore, its effect on protein synthesis isreversible, which allows studies of the effect of inhibitions at particular periodsof synthesis in relation to morphogenesis. Since a pulse period of two hours orless of pactamycin does not effect RNA synthesis, a clear distinction betweeneffects referable to translation rather than transcription can be made.

Failures of morphogenesis due to precisely timed pulses of pactamycin fall ina serial order whose regular sequence is predictable and closely related to asimilar serial order elucidated by inhibition of RNA synthesis with actinomycin D.

It is this predictable sequence of morphogenetic failures due to precisely timedadditions of pactamycin to the incubation medium of the embryos that we wishto stress at this time in correlation with our previous data on the morphogeneticdefects related to RNA synthesis inhibition (Wilde & Crawford, 1966, 1968).

Development to the high blastula (stage 9-10) in Fundulus is independent ofcontemporary RNA synthesis on zygotic templates although in the zygote thereis initiated synthesis of RNA meaningful for the post-blastula period beginningwithin minutes following fertilization. The data for Fundulus are consistent withthe findings in a broad range of embryonic systems (Davidson, 1968). Theconclusion generally held is that genomic transcription of informational mole-cules required for cleavage occurs prior to fertilization and thus on maternaltemplates. This would include active templates for proteins concerned withcleavage spindles, chromosome replication and increment of cell surface. Thishas recently been established in echinoderms (Raff, Colot, Selvig & Gross,1972).

Inhibition of protein synthesis through administration of pactamycin im-mediately upon fertilization aborts the first cleavage. If the pulse initiation isdelayed an effect decreasing in intensity is observed up to a pulse initiation timeof 20 min following fertilization. In the latter circumstances, sparing the first10 to 20 min permits some zygotes to undergo two or three cleavages. Thereforeduring this time anticipatory syntheses of cleavage associated proteins are takingplace presumably upon maternal templates. Anticipatory syntheses appear tobe required for cleavages two or three cycles in the future. Therefore, an ongoingprotein synthesis is required in Fundulus for normal development in the cleavage


Table 6. Comparison of actinomycin D andpactamycin effectson Fundulus morphogenesis*

InhibitorInitiation , A- — — —

timej Effect on morphogenesis Actinomycin D Pactamycin

0-11-22-33-44-55-10

10-3030-60

Prevention of early cleavageDefective blastulaDefective gastrulationDefective axiationAnencephalyLess severe anencephalyMicrocephalyNormal 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, cleavageto the high blastula is normal in form and in timing.

We have previously reported (Wilde & Crawford, 1966) a serial order ofdefects in morphogenesis of high predictability dependent strictly upon the timeof initiation of actinomycin D inhibition of RNA synthesis. The data presentedin this paper demonstrate the similarity of morphogenetic defects beyond thehigh blastula conferred by pulses of pactamycin and actinomycin D. Thesecorrelations are shown in Table 6. The morphogenetic defects are expressedmorphologically 40 or more hours after the cessation of the inhibitory pulsewhich led to the defect. While protein synthesis as a general phenomenon ismuch more rapidly restored following pulse termination, the morphogeneticdefects are permanent.

We are thus drawn to the conclusion that specific protein syntheses, requiredfor normal morphogenesis beyond the blastula, are initiated prior to the secondminute following fertilization and thus upon zygotic templates (or alternativelyon 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 ter-mination of the pulse or shortly thereafter since protein synthesis as measuredby 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 andhistone synthesis, to name the most obvious. It would appear therefore that theconcurrent protein synthesis required for post-blastula morphogenesis takesplace at a very low level. If informational RNA is presumed to be attached toappropriate ribosomal configurations and the presence of potentially functional


ribosomal units is assumed, upon relief of the inhibition, why is subsequentmorphogenesis disturbed? Perhaps pactamycin firmly enters the functional unitand renders it inactive on a relatively permanent basis. Consequently, since aserial order of RNA synthesis essential to post-blastula morphogenesis has beendemonstrated to be time dependent, the essential first morphogenetically mean-ingful proteins are never given the morphogenetically correct, temporal oppor-tunity to be synthesized, their templates having been passed by in time and,perhaps, degraded. Relief of the inhibition may allow for all the essential proteinsyntheses, certain of which are now out of proper sequence and relationship tothe ongoing development. Under these conditions they cannot play their normalmorphogenetic role.

It follows, in the major axial systems studied here in Fundulus, that earlysyntheses are successionally dependent upon antecedent ones. Only after thefifth minute is there any expression of reasonably normal morphogenesis of onepart (viz. gut) while another part (the brain) remains utterly defective. Morpho-genetically meaningful macromolecular syntheses appear to fall into a ramifyingscheme which is initially one tract prior to branching. Organogenesis of aparticular structure would be inhibited by failure of antecedent syntheses at abranch point. Under such circumstances, after the fifth minute, inhibition alongone ramus would lead to the failure of morphogenesis of subsequent dependentdevelopment while along an uninhibited branch morphogenesis would continuemore or less normally. Such a scheme however, must be broad enough concep-tually to include connecting links at varying levels of organogenesis.

These data are consistent with the conclusion that morphogenesis in Fundulusis under genomic control and that the control functions through the wellestablished mechanisms of transcription and translation. They further indicatethat the macromolecular chemistry of morphogenesis begins within seconds offertilization at least as expressed in the primary and major morphogeneticactivities of the zygote. The expression of genomic control as reflected in thetime dependent serial order of developmental failures here analyzed indicatesthat morphogenetic chemistry proceeds rapidly and stepwise. Failure at earlysteps causes abnormal orientation of any further development.

Yet it is of intense interest that development does in fact continue, carryingthe immutable defects, notochord without nervous system, nervous systemwithout somites, etc. The morphogenetic program entered into at fertilizationbehaves as though all cells and their progeny were committed and cognizantof time flow and position; only those cells which were the open targets of theinhibitor during, and only during, the pulse failed in morphogenetic commit-ment and behaviour.

It has long been considered that morphogenesis is initiated at gastrulation viainductive processes. We wish to emphasize that the classic phenomenology is,in Fundulus, preceded by 40 h of essential macromolecular synthesis withoutwhich morphogenesis is aborted. Indeed the most important initiating and

25 EM B 29


controlling events appear to be confined to the period immediately followingfertilization and well within the first cleavage period while the zygote is a singlecell.

The specific protein syntheses characteristic of cellular differentiation areexpressed in many of the terata discussed here. These are also presumably undergenomic control. However, the transcription and translation processes for cellulardifferentiation appear to be initiated later in embryogenesis beyond the periodof morphogenetic determination. The relationship of primary morphogeneticprocesses to the chemistry of cellular differentiation remains to be explored.

The authors wish to thank Mrs Diane Zucker and Mrs Michele Koppelman for theirexcellent technical assistance. Gift of pactamycin from the Upjohn Co. was very muchappreciated.

The work reported here was supported by N.S.F. Grant GB-6766 and an NSF Grant tothe Mount Desert Island Biological Laboratory GB-28139.

REFERENCES

ARMSTRONG, P. B. & CHILD, J. S. (1965). Stages in the normal development of Fundulusheteroclitus. Biol. Bull. mar. biol. Lab., Woods Hole 128, 143-168.

COHEN, L. B., GOLDBERG, I. H. & HERNER, A. E. (1969). Inhibition by pactamycin of theinitiation of protein synthesis. Effect on the 30S ribosomal subunit. Biochemistry, N.Y.8, 1327-1344.

COHEN, L. B., HERNER, A. E. & GOLDBERG, I. H. (1969). Inhibition by pactamycin of theinitiation of protein synthesis. Binding of N-acetylphenylalanyl transfer ribonucleic acidand polyuridylic acid to ribosomes. Biochemistry N. Y. 8, 1312-1326.

COLOMBO, B., FELICETTI, L. & BAGLIONI, C. (1966). Inhibition of protein synthesis in reticu-locytes by antibiotics. I. Effects of polysomes. Biochim. biophys. Ada 119, 109-119.

CRAWFORD, R. B. & WILDE, C. E., JR. (1966). Cellular differentiation in the anamniota.IV. Relation between RNA synthesis and aerobic metabolism in Fundulus heteroclitusembryos. Expl Cell Res. 44, 489-497.

DAVIDSON, E. H. (1968). Gene Activity in Early Development. New York: Academic Press.FELICETTI, L., COLOMBO, B. & BAGLIONI, C. (1966). Inhibition of protein synthesis in reticu-

locytes by antibiotics. II. The site of action of cycloheximide, streptovitacin A and pacta-mycin. Biochim. biophys. Ada 119, 120-129.

ITZHAKI, R. F. & GILL, D. M. (1964). A micro-biuret method for estimating proteins. Analyt.Biochem. 9, 401-410.

KAFIANI, C. (1970). Genome transcription in fish development. Adv. Morphog. 8, 209-284.NEW, D. A. T. (1966). The Culture of Vertebrate Embryos. New York: Academic Press.OPPENHEIMER, J. M. (1937). The normal stages of Fundulus heteroclitus. Anat. Rec. 68, 1-15.RAFF, R. A., COLOT, H. V., SELVIG, S. E. & GROSS, P. R. (1972). Oogenetic origin of mes-

senger RNA for embryonic synthesis of microtubule proteins. Nature, Loud. 235, 211-214.TYLER, A. & TYLER, B. S. (1970). Informational macromolecules and differentiation. In Cell

Differentiation (ed. O. A. Scheide & J. de Vellis), Chap. II, 4, pp. 42-118. New York:Van Nostrand-Reinhold.

WILDE, C. E., JR. & CRAWFORD, R. B. (1966). Cellular differentiation in the anamniota.III. Effects of Actinomycin D and cyanide on the morphogenesis of Fundulus. Expl CellRes. 44, 471-488.

WILDE, C. E., JR. & CRAWFORD, R. B. (1968). Epithelial mesenchymal interactions in thelower vertebrates. In Epithelial-Mesenchymal Interactions (ed. R. Fleishmayer), Chap. 6,pp. 98-113. Baltimore: Williams & Wilkins Co.

{Received 18 May 1972, revised 30 August 1972)

morphogenetic disturbances from timed inhibitions of ...fundulus heteroclitus (linnaeus) was...

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