The Egg Cortex Problem as Seen Through the Squid Eye

AMER. ZOOL.. 16:421-446 (1976).

The Egg Cortex Problem as Seen Through the Squid Eye

JOHN M. ARNOLD

AND

Lois D. WILLIAMS-ARNOLD

Kewalo Marine Laboratory of the Pacific Biomedical Research Center,University of Hawaii, Honolulu, Hawaii 96813

andMarine Biological Laboratory, Woods Hole, Massachusetts 02543

SYNOPSIS In many spiralian embryos it has been possible to demonstrate that embryonicdevelopment is partially controlled by cytoplasmic factors located at or in the surface of thefertilized egg and cleaving embryo. In the embryo of the squid Loligo pealei, a pattern ofdevelopmental information can be demonstrated to exist at the surface, or the egg cortex, ofthe fertilized but uncleaved embryo. The informational pattern apparently is released oractivated during the time of the cytoplasmic streaming which forms the blastodisc. Eventu-ally this developmental informational pattern is imposed upon the blastoderm cells thatcome to cover organ-specific regions of the yolk syncytium which was derived from the eggcortex. Ultrastructural studies demonstrate many intercellular connections between theyolk syncytium and the blastoderm and between the cells of the blastoderm itself. Duringoogenesis there are regional differences in the follicular syncytium which suggests that thepattern of developmental information may arise in the ovary and be retained in a latent stateuntil triggered by fertilization.

INTRODUCTION

The role of cytoplasmic factors in thecontrol of development and differentiationhas been amply demonstrated in a largenumber of spiralian species and it wouldnot be possible to mention all the pertinentwork in the space available. The otherspeakers in this symposium have contrib-uted greatly to our current knowledge ofthe segregation of development directingcytoplasmic factors during cleavage andtheir papers undoubtedly offer a broader

Since this paper is a summary of many years re-search, it is not possible to mention all of the individu-als who have contributed to the experiments and ideasincluded here. However, a few people have made re-cent contributions which cannot be overlooked. Dr. M.G. Hadfield has offered many helpful suggestions. Mr.T. Joiner Cartwright, Jr., and Dr. Beverly H.Bergstrom have provided some of the electron micro-graphs. Mr. Carl T. Singley, Jr., has read and im-proved this manuscript, and Ms. Frances Horiuchi hasdone an excellent job preparing it. To these peopleand many others not mentioned, we express our sin-cere appreciation. This work has been supported byseveral grants from the National Institutes of Healthand the National Science Foundation of the UnitedStates.

and better survey of the field than is possi-ble here. However, to set forth the prob-lem, the work of E. B. Wilson (1904a,*) onDentalium will be used as an example. Byremoval of the polar lobe in progressivelyolder cleavage stages, Wilson was able todemonstrate that not only did cytoplasmicfactors control differentiation of specificlarval structures, but further that thesegregation of these factors was progressivein time. Operations on older and older em-bryos produced narrower and narrower"lobeless" deficiency syndromes. In normaldevelopment the polar lobe comes to con-tain cytoplasmic structures and inclusionsquantitatively different than the rest of theembryo. It is tempting to assume that thesenatural cytoplasmic segregations separatespecific precursor substances which ulti-mately code for specific embryonic or larvalorgans. However, rearrangements of thesespecial cytoplasms by centrifugation hasdemonstrated that differentiation is con-trolled by regionalized factors not directlyassociated with easily displaced observablecytoplasmic differences (Verdonk, 1968;Clement, 1968). Therefore, a non-moving

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422 JOHN M. ARNOLD AND LOIS D. WILLIAMS-ARNOLD

component has been implicated and theegg surface and its underlying cytoplasmhas come under suspicion. This specializedsurface structure has been called the eggcortex by many authors and has been de-Fined functionally as "the pattern of de-velopmental information that resides in theegg surface which is not displaced by oo-plasmic streaming or other displacement ofthe cytoplasm" (Arnold, 1968). The pur-pose of this paper is to discuss and sum-marize the role of the egg cortex in thecontrol of development and differentiationof the cephalopod Loligo pealei. Essentiallythere are four questions to consider whendiscussing possible cortical control of em-bryonic development and differentiation.These are: 1) Can a pattern of developmen-tal information be demonstrated and if sowhat is its structural and biochemical na-ture? 2) What triggers an informationalpattern to go from an inactive latent state toan active state? 3) How does the informa-tional pattern express itself during organ-ogenesis? and 4) How and where does theinformational pattern originate? Each ofthese questions will be discussed in turn butpresently only partial answers are possibleand gaps in our knowledge of cephalopoddevelopment will be painfully obvious.

NORMAL DEVELOPMENT OF THE SQUID

Although the normal development ofLoligo has recently been described (Arnold,1971; Meister, 1972; Fioroni and Meister,1974; Arnold et al, 1974), a brief descrip-tion of normal ontogeny is necessary herefor the convenience of the reader and tomake comparisons with gastropod de-velopment. This survey will mainly em-phasize early development and the readeris referred to the above papers for details oforganogenesis.

Cleavage, gastrulation and cellulation

In Loligo pealei the egg is large (1.6 X 1mm), telolecithal, and ovate in shape. It isenclosed in a clear chorion which has a mi-cropyle at the animal apex of the egg. Dur-ing egg laying by the female, several scores

of eggs are passed down the oviduct andencased in a common fold of egg jelly whichis then twisted into a spiral and enclosed inseveral layers of egg jelly. As this egg stringis passed out of the female's funnel, sper-matozoa from the spermatophores depos-ited by the male during copulation pene-trate the egg jelly, accumulate at the micro-pyle, and fertilize the egg (Arnold, 1962;Tardent, 1962; Fields, 1965). Therefore,all of the embryos enclosed in any given eggstring are siblings and are fertilized at thesame time so early development is syn-chronous. Polar body formation occursafter the sperm enters the egg and a blas-todisc of clear cytoplasm is formed by pro-gressive migration of cytoplasm from thethin layer between the yolk and egg surface.Some cytoplasm remains in association withthe plasma membrane and this mem-brane-cytoplasm complex has been struc-turally denned as the egg cortex (Arnold,1968).

Cleavage occurs in the blastodisc and, atfirst, is incomplete because of the massiveyolk. The cleavage pattern is markedlybilateral with the first furrow separatingthe future right and left halves and thesecond furrow separating the future em-bryonic anterior and posterior. Thirdcleavage in typical spiralians is charac-terized by the angle of the furrows "violat-ing" Balfour's rule which states that cleav-age occurs at right angles to the previousfurrow. In a typical spiralian, the third fur-row is formed at an angle to the perpen-dicular so that the resulting animal blasto-meres (= micro meres) lie in grooves be-tween the usually larger vegetal blasto-meres (= macromeres). In the cephalopods(which because of the massive amount ofyolk are essentially two dimensional in earlycleavage), there is only a remnant of thiscleavage angle change (Fig. la) (Arnold,1971; Arnolds al, 1974; Naef, 1928; De-Leo, 1972). The posterior third furrowparallels the first furrow, and therefore, isat right angles to the second furrow. How-ever, the anterior third furrow occurs at anangle of 30° to the perpendicular thus caus-ing the inner anterior blastomeres to belarger than the inner posterior blasto-meres. Although the angle of the third

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EGG CORTEX OF THE SQUID 423

cleavage furrow in relation to the first andsecond cleavage plane is a tenuous threadto attempt to connect the cephalopods tothe spiralians, there are good taxonomicreasons to relate the cephalopods to themollusca.

This cleavage pattern seems to be pre-determined to a certain extent because eggspulsed with 0.2 /xg of Cytochalasin B (CB)for 10 minutes, one hour before first cleav-age, display an "equalized" pattern of fur-rows when third cleavage occurs (Arnoldand Williams-Arnold, 1974). In thesetreated eggs, the posterior furrows occur atvarying angles to the second furrows andare not parallel to the first. Since the CBpulse occurred three hours before the ap-pearance of the third furrow (and approx-imately 2 hours and 40 minutes before themitotic spindle determined the furrow po-sition), it would seem that some CB sensi-tive prepattern of nuclear positioningexisted in the precleavage blastoderm.

After completion of the second furrow,the bases of the first and second furrow fuseand continue to contract so they cut hori-zontally between the cytoplasm and theyolk as an undercutting furrow. Thus, theblastomeres at the four cell stage are incontinuity at the edge of the blastodisc butbecome partially separated from the yolkbeneath them.

Fourth cleavage separates the blasto-derm into two populations of cells: thecentral cells which are not in cytoplasmiccontinuity with each other and theperipheral cells which remain attached tothe egg proper at their distal edges. Simi-larly, fifth cleavage divides the inner cen-tral daughter cells free from their peri-pheral continuity so two populations ofcells are established; a central group whichis cytoplasmically independent and aperipheral syncytial group surroundingthe developing blastoderm. Subsequentcleavages continue to add to the growingcentral blastoderm while the peripheralcells expand to form a syncytial ring beyondthe margin of the blastoderm (Fig. la,b).

It is tempting to make homologies withother spiralians and compare the ceph-alopod meroblastic cleavage patternwith holoblastic cleavage found elsewhere.

Unfortunately, this can be carried to anextreme and the term "micromeres" hasbeen used to designate the central blasto-derm cells as distinct from the "macro-meres" of the peripheral syncytial cells(Fioroni and Meister, 1974). Since the cen-tral blastomeres are not derived untilfourth cleavage, they are not strictly com-parable to the first quartet (see Costello andHenley, 1976). Attempts at making celllineages are mainly exercises in numerol-ogy since the number of cell divisions be-fore organogenesis begins obviates follow-ing individual cells to their definitive fate(DeLeo, 1972). However, with these cau-tions in mind, it is interesting to comparecephalopod cleavage with that of a moretypical spirally cleaving egg. The first twocleavages are incomplete resulting in anembryo in which the "ABCD" remains asone cell. Third cleavage is also incompletebut is unequal as noted above. Instead of apopulation of cells made up of several quar-tets situated upon four macromeres, ablastoderm is established which is pe-ripherally surrounded by a syncytialring. In some cephalopods the cells of theperipheral ring are quite distinct trianglesand are called blastocones (Vialleton,1888). These blastocones contribute cellsfrom their base to the growing blastodermbut themselves become a highly special-ized population which remains syncytialthroughout embryonic development andhas special significance in differentiation.Thus, the similarity of a cephalopod em-bryo in early cleavage to a gastropod em-bryo just before the onset of epiboly is evi-dent.

Gastrulation is also modified by theamount of yolk and has been the subject ofsome speculation but little investigation.The description which follows here is fromas yet unpublished work of Singley (per-sonal communication). After approxi-mately the 9th cleavage (stage 9, Arnold,1965a), the cells three cellular rows in fromthe edge of the blastoderm begin to extendperipherally over the row of cells distal tothem (Fig. lb). Ruffled membranes areevident at the edge of these extensions andthis expansion is Cytochalasin B sensitive.Once the overlying cells have completely

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covered the peripheral row, the now cov-ered cells undergo a marked increase inmitotic rate and a three-layered embryo isformed. The outer layer is composed ofindividual cells which divide perpendicularto the surface of the embryo. The middlecells form a ring just beneath the margin ofthe blastoderm and divide apparently inrandom directions. The syncytial, periph-eral cells undergo division and form atransitory furrow perpendicular to theanimal-vegetal axis. This furrow firstforms, then disappears, leaving a doublering of nuclei in a syncytium beyond themargin of the blastoderm. These nuclei di-vide at least one more time and give rise tothe yolk syncytium (=yolk epithelium). At-tempts have been made to force the termsectoderm, mesoderm and endoderm onthese three components of the cephalopodembryo but, since the yolk syncytium doesnot become incorporated into the definitiveanimal, parts of the nervous system are de-rived from "mesoderm," and the origin ofthe midgut is still in question, it seemssenseless to push homologies to the break-ing point. Cephalopods have been develop-ing for more than 180 million years ignor-ant of the names of their germinal layersand more basic questions are better askedof them.

Once the three basic layers have beenestablished, the remaining yolk surface

(formerly the egg surface), becomes cov-ered by the blastoderm. The middle layerof cells expands toward the animal pole andeventually completely underlies the outerlayer of cells. In so doing they form a singlesheet from the formerly randomly ar-ranged cellular ring. The margin of theblastoderm expands to cover the yolk syn-cytium so that the original egg surface becomescovered with blastoderm cells but is not greatlydisplaced. This is analogous to epiboly infishes but can be inhibited with colchicineand is, in part, due to mitosis within theblastoderm and at its edges.

When the expanding blastoderm reachesthe future margin of the yolk sac, the mid-dle layer of the blastoderm becomes diffuseand the outer cells are more flattened thanin the future organogenic region. Eventu-ally, a hemal space develops between theouter cells and the yolk syncytium and theexternal yolk sac develops in this position.Muscle cells develop, transversing thehemal space and their contraction is syn-chronized to circulate the blood. The cellsof the external yolk sac develop cilia (stage17, Arnold, 1965a) and the external yolksac undoubtedly also functions as a respi-ratory organ.

Organogenesis

Organogenesis in the cephalopods is

FIG. 1. Normal developmental stages of Loligopealeiasvisualized with the scanning electron microscope,la. Blastomeres at 16 cells (stage 7). The polar bodies(pb) are to the right of the first cleavage furrow and asmall gap is present at the junction of the first andsecond furrows. The inner fourth furrows divide thetwo daughter cells while the outer fourth furrows ex-tend only to the edge of the blastodisc, thus the outercells are continuous at their extreme outer edges. Inthis way two populations of cells are established: Thoseindividual cells of the central blastoderm and thoseperipheral cells which remain syncytial. xllO.lb. Blastoderm at early stage 10. The overlayeringring of cells (arrow) is expanding peripherally to coverthe neighboring cells. The covered cells then undergorapid divisions and build up a population of cells be-tween the yolk syncytium and the newly formed outercellular layer. XllO.lc. Stage 18 dorsal view showing organ formationfollowing cellulation of the yolk mass. The vegetal halfof the embryo becomes ciliated and forms the externalyolk sac. The eyes are prominent (e) and the mouth(mo) is forming. The mantle (m) has begun to expand

outward and funnel folds are appearing above theeyes near the mantle (ff) (arrow). The arm primordiaare evident in the equatiorial region. x40.Id. Stage 22 ventral view. The organ primordia area ismuch more prominent and distinct. A hemal space(hs) has developed in the external yolk sac. The outercellular covering has been reflected in the area indi-cated. The gills (g), anal papilla (a), statocysts (s), andfins can be distinguished. Other labels as in lc. x65.le. Ventral view of a stage 25 embryo. The funnelfolds have fused to make the siphon (si) and suckershave appeared on the arms. The mantle has growndownward to cover the visceral organs. x50If. Dorsal view of stage 25. The eyes form prominentprojections on the head.lg. Lateral view at stage 27. The definitive cornea has

just begun to grow over the eye. x60.lh. Dorsal view of stage 29. Note the prominent linesof ciliated cells on the back. The head has cilia in singlelines and in single cell patches. X40.li. Post-hatching juvenile. Note the epithelium of themantle is being sluffed but the ciliature of the head isintact. x40.

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unique in that development is essentiallydirect to form a miniature adult, occurs onthe surface of a central yolk mass, and re-flects the long evolutionary isolation fromthe rest of the Mollusca (Fioroni, 1974).What follows here is a very brief survey ofsurface features of development to ac-quaint the reader with organogenesisenough to make the following experimentsinterpretable. For further details ofdevelopment, the reader is referred to therecent papers of Arnold (1971), Meister(1972), Fioroni and Meister (1974), or Ar-nold et al. (1974). The developmental stagesreferred to here are those of Arnold(1965a).

As the blastoderm spreads to completelycover the entire yolk mass, the organs beginto form from the cells of the blastoderm.The first organ to appear is the shell gland,which at stage 16 forms at the animal poleas a localized thickening. The edges of thisthickening elevate and constrict to form aring-shaped flattened vesicle. During laterdevelopment of the mantle, the cartilagen-ous pen is formed within the "shell gland."

At about stage 17 and 18, the majororgan primordia appear (Fig. lc, d). Theexternal yolk sac, described above, occupiesthe vegetal half of the embryo of Loligopealei. In other cephalopods it may be im-mense with the embryonic rudiment occur-ring as a small patch at the animal pole (e.g.,Sepia, Lemaire, 1970) or almost non-existent (e.g., Todarodes, Hamabe, 1962).The eyes, statocysts, arms, mantle, mouth,and funnel folds appear as thickenings inthe blastoderm and at stage 18 and 19 canbe seen as distinct organ primordia. Thearms originate as two thickened bands atthe margin of the external yolk sac and latersegregate into the eight arm and two tenta-cle primordia (Fig. lc). They elongate, de-velop suckers and encircle the constrictingneck of the external yolk sac (Fig. Id). Thefunnel folds appear as two paired elongatethickenings on the future ventral surface(Fig. Id).These thickenings increase in size,fuse together, and eventually meet at themidline to form the tubular siphon and as-sociated musculature (Fig. le). Between theeyes and the funnel folds, the statocysts ap-pear as slight depressions which invaginateto form a closed vesicle (Fig. Id). The

statolith is formed by fusion of droplets ofGolgi-produced substance which is secretedinto the lumen of the statocyst. The gillsappear as paired protuberancesjust medialto the posterior funnel folds and branchand ramify as the circulatory vessels de-velop within them. The gut arises on thefuture functional ventral surface as an iso-lated mouth invagination and on the dorsalsurface as the anal papilla (Fig. lc, d) whichhas a similar invagination, the hind gutprimordium. As development proceeds,the two ends of the gut meet via the midgutwhich appears to have an independent ori-gin (Boletzky, 1967, 1970).

The mantle first appears as a ring whichgrows outward then toward the eyes tocover the developing viscera in a thick mus-cular cup. At about stage 17 cilia appear atthe margin of the mantle and develop into adistinct pattern which circulates the intra-chorionic fluid over the embryo. At hatch-ing the surface epithelium is sluffed(Fioroni, 1963) (Fig. li). During later stagesof development, the yolk is actively trans-ferred from the external yolk sac into theinternal yolk sac so at hatching only a smallremnant is left. The external yolk sac rudi-ment is dropped by the embryo; thus themouth is made available for feeding.Hatching is accomplished by the digestiveaction of the Hoyle organ which is appliedto the inside of the chorion and opens ahole the shape of the organ.

Since this paper deals with the visualorgan as a model system of differentiation,the development of the eye will be dealtwith in some detail. The eyes originate asthickened primordia on either side of theembryo (Fig. lc). Because they are paired, itis possible to use one eye for experimentalanalysis and have the other serve as a con-trol. At the edges of the eye primordia, acircular ridge develops which comes tooverlap the future retina and by graduallyextending centripetally from the edges itforms the flattened optic vesicle (Fig. 2).Whether or not the formation of the flat-tened optic vesicle can be called an invagi-nation or not is a question of semantic sig-nificance only (Marthy, 1973). Graduallythe optic vesicle becomes spheroidal andthe proximal portion differentiates into theretina while the distal portion gives rise to

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FIG. 2. Development of the cephalopod eye. See text Lentigenic cells, Ir = Iris, Or = Orbit, G =for details. R = retina, L = lens, Op = optic vesicle, C = ganglion,cornea, PI = pigment layer, Rd = rhabdome, Lg =

2

optic

the lens and iris. The future retina under-goes rapid mitosis at first and builds up alayer of evenly distributed cells. The rhab-domes develop on the inside of the opticvesicle and each sends a single neurofibrilback past the developing pigment and sup-porting cells to the ganglion. The lens arisesfrom the fusion of cellular processes whichare sent out of a group of large specializedcells; the lentigenic cells (Arnold, 1967).Within the resulting onion bulb-like lensprimordium, the definitive lens material issynthesized from polysomes and Golgi-produced vesicles in association with theplasma membrane. Eventually the cyto-plasmic portions of the lens are entirelyreplaced by the inflexible, nonliving, lens

material except for a few surface layers. Atapproximately stage 27, the definitivecornea appears as a fold of tissue whichgrows over the eye. Thus the eyeball canmove freely about inside the orbit while thecornea remains fixed. The retina apparent-ly induces the formation of the optic gan-glion (Ranzi, 1931; Arnold 19656). The de-veloping eye and optic ganglion make up aconsiderable portion of the head. Thebrain develops by fusion of the optic, pedal,cerebral, visceral, and branchial ganglia.

THE EGG CORTEX AS A SITE OF DEVELOPMENTALINFORMATION

Because of its large size, which facilitates

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manipulation, and the bilateral cleavagepattern, which facilitates orientation of theearly embryo, the squid embryo is ideallysuited for studies on the egg cortex. It hasbeen possible to demonstrate that there is apattern of developmental information res-ident inthe egg surface of the squid embryoand that this information is positionally de-termined very early in development. Whatfollows here is a brief summary of the evi-dence for cortical control of cephalopoddevelopment. For further details, see Ar-nold (1968), Arnold and Williams-Arnold(1974), and/or Boletzky (1970).

If the embryo is removed from the eggjelly during first cleavage, it is possible toorient it so the future left and right sidesand the embryonic anterior and posteriorare evident. By using a light microbeamapparatus, it has been possible to regionallyirradiate the superequatorial region of thetwo cell embryo. This "target region " is

approximately 0.75 mm from the blastodiscand nuclei. Subsequent development(cleavage, gastrulation, and cellulation ofthe yolk mass) appears to be normal untilthe beginning of organogenesis. Althoughthe "target region" is eventually coveredwith cells several layers thick, or-ganogenesis either does not occur or par-tial, reduced, or otherwise abnormal or-gans are formed (see Fig. 3). Using ul-traviolet light in the range of 260 nm pro-duced a similar effect although the lengthof time of irradiation must be increased. S.Freeman (personal communication) hasfound similar results with ultraviolet ir-radiation of Ilyanassa embryos and pro-duced abnormalities correlated with theposition of irradiation of the polar lobe.Dohman and Verdonk (1974) have shownin Bythinia that the polar lobe contains avesiculate "vegetal body" which stains asRNA. Removal of the polar lobe causes

FIG. 3. (a) Embryo irradiated in the equatorial regionduring first cleavage. The second, third, and fourtharms of the left side are missing and the eye is reduced.The arrow indicates the position of the histological

* V

section shown in (b). X50. (b) The irradiated area wascovered by cells but no histogenesis took place. Repro-duced by permission of Academic Press.

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organ deficiencies similar to those found inother gastropod embryos (Cather and Ver-donk, 1974).

It is possible to demonstrate that a corti-cal pattern of developmental informationexists even later in cephalopod develop-ment (stage 10 and 11). By ligating off smallpieces of uncellulated egg surface, it waspossible to correlate the region removedspecifically with the organ deficiencies pro-duced later in development (Fig. 4) (Ar-nold, 1968; Boletzky, 1970). If the ligaturetransects the lower vegetal pole region only(future external yolk sac) the resultant em-bryos are reduced in size by the loss of yolkvolume but tend to develop as smaller butcomplete organisms. However, if the liga-ture is placed in the animal region whichtopographically produces the embryonicorgans, specific deficiencies arise whichcorrelate with the position of the cortexremoved (Fig. 4). Table 1 shows the cor-relation of position of the ligation andorgan deficiencies. Marthy (1975) ligatedseven embryos of two different speciesbelow the future organogenetic region andobtained results similar to those shown inFigure 4b. However, he interpreted his re-sults to mean that the developmental in-formation necessary for differentiation re-sided solely in the cells of the blastoderm. Itshould be kept in mind that in the irradia-tion experiments and in large part in theligation experiments the affected area doesnot include nuclei yet the deficiencies whichresult are positionally specific.

There is the possibility that specificcytoplasmic structures as opposed to corti-cal factors might be regionally regulatingorganogenesis. The cytoplasmic streamingwhich occurs to form the blastodisc wouldargue against a mobile cytoplasmic factorbecause the cytoplasmic layer between theplasma membrane and the underlying yolkis considerably reduced in thickness as theblastodisc forms (see, however, cytochala-sin effects reported below). The cytoplasmcan also be experimentally displaced by lowspeed centrifugation at the time of meiosis.The cytoplasm is displaced to the centri-petal pole (one side of the ovate embryo)and as cleavage follows the cells on that sidetend to be larger. At organogenesis the or-

gans on the centripetal pole on the egg tendto be larger while those on the centrifugalpole are reduced in size. However, theplacement of the organs is approximatelynormal (Fig. 5).

ACTIVATION OF THE CORTICAL INFORMATION

Treatment of early embryos with thedrug cytochalasin B offers further insightinto the mechanisms of cortical influenceon differentiation. If the embryo in pre-cleavage or early cleavage is regionallytreated for 10 minutes by applying agarsoaked in a 2.0 Mg/ml solution of cytochala-sin B to one portion of the future organo-genic area, positionally related effects areobvious during organogenesis (Fig. 6). Itshould be noted that this regional treat-ment is well away from the blastodisc orblastoderm and if properly applied doesnot interrupt cytokinesis or cause other ap-parent disturbances. Cellulation appears toproceed normally and organogenesis in un-treated regions occurs in synchrony withthe controls. However, in the treated reg-ions organogenesis is inhibited and eitherpartial organs are formed or no histo-genesis is evident (Fig. 6b). Marthy (1973)argues that this is an effect caused by dis-ruption of the nutritional function of theyolk syncytium. If this were the case, how-ever, the ligation experiments describedabove would still indicate that there is reg-ional specificity in the cortex which directsthe overlying cells along particular paths ofdifferentiation.

Furthermore, pulse treatments of wholeembryos with cytochalasin B at low dosages(0.2 fig/ml for 10 min) cause effects onorganogenesis correlated with the time oftreatment (Fig. 7). If embryos were pulsedbefore or about 1st polar body formationthe drug produced relatively minor effects.If treated after gastrulation when theblastoderm was expanding over the eggsurface, (St. 11, 13, 15) there were negligi-ble effects on organogenesis. However, ifthe embryos were pulsed during the periodof the major cytoplasmic streaming whichforms the blastodisc, organogenesis was se-verely affected. These embryos displayeddisplaced or missing organs or, in severe

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EGG CORTEX OF THE SQUID 4S1

FIG. 5. Results of centrifugation of the fertilized em-bryo after first polar body formation, (a) The cleavagefurrows are evident and the nuclei can be seen, (b)Embryo resulting from similar centrifugation experi-

cases, lacked organ differentiation entirely(see Arnold and Williams-Arnold, 1974 fordetails). If the cytochalasin treatment wasaffecting the nutritive function of the yolksyncytium, then this temporal sensitivitywould seem highly unlikely; rather an in-creasing sensitivity might be expected asthe time of treatment approached the onsetof organogenesis. It should be stressed thatalthough cleavage patterns tend to be ab-normal and that the early blastoderm cellstend to be more equal in size than in con-trols, cellulation of the yolk surface appearsnormal and after the initial delay of cleav-

ment. The organ primordia are normally placed butthose on the centrifugal side (cytoplasm poor side) aregreatly reduced. Reproduced by permission ofAcademic Press. x50.

age, development proceeds normally untilorganogenesis.

There are two periods of sensitivity tocytochalasin pulses that do cause im-mediate effects: formation of the under-cutting furrows and gastrulation. Disrup-tion of the undercutting furrows preventsformation of the subcellular space (=blas-tocoel) and gastrulation is prevented.Treatment just before gastrulation disruptsthe overlayering of the cellular ring andthus prevents gastrulation. These embryoshave obvious mechanical problems whichcan be distinguished from the longer term

FIG. 4. Results of ligation experiments, (a) Embryoconsists of essentially a mantle, eyes, and anterior or-gans. The arms and external yolk sac are missing, (b)Embryo ligated further down has arms but the exter-nal yolk sac is missing, (c) Embryo ligated horizontallyabout two-thirds of the way from the animal pole. Notethat the external yolk sac is reduced in size. (d,e) Re-duction of the yolk sac by horizontal ligations through

the vegetal half of the embryo, (f) Diagonal ligationresulting in unilateral reduction of some anterior or-gans, (g) Loss of eye and three arms by diagonal liga-tion. (h) Reduction of mantle and loss of the anteriororgans by ligation. (i) Control embryo, symbols as inFigure 1. Reproduced by permission of AcademicPress. X35.

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TABLE IRESULTS OF THE LI CATION EXPFRIHF.NTS

organs p r e s e n t

Fmbryo LigatureNumber Position

3

4

5

6

7

8

9

in

11

14

15

16

17

18

19

20

21

22

23

24

%-Q-

0

6

t0

ft eve

-

-

-

4

01

a

3

4

2

1

4

-

4

4

-

ft fun

lei folds

-

•

4

-

-

4

-

4

oo:yst

-

-

-

-

>cyst

4

-

-

4

-

runnel folds

-

4

-

•

4

2

4

•

'

4

4

4

-

*

4

at

*

•

4

4

4

4

4

4

4

0U sac

•

4

4

4

4

4

4

4

4

4

4

4

4

0

IT

-

4

4

4

4

•

4

4

4

4

4

Results

esentiallv normal

•antle reduced,someant. organs missing

only arms and yolk sac

no volk sacno arms

greatly reducedyolk sac

reduced volksac by 1/2

reduced yolk sac

reduced volk sac

eve, 2 arms, otocvst

nantle reduced, someant. organs missing

area between mantleand mouth reduced

eve, otocyst, 1 a nmissing

area between eveand otocvst reduced

missing 3 arms, otocvst

ant. organs missingeye reduced

J arms, eye missing

mantle reducedant. organs missing

eve Teduced to1/2 size

reduced mantle

reduced mantle

azvs, ant. organsroissinp, eve andmantle reduced

.•>

-5

3

i

4a

4b

4c

4d

4c

4f

4K

4h

subtotal 24 live and interpretable embryos1 live but no organ development

IS arrested in development8 lysed after first photoRraph

12 cells sloughed off

19 died of unknown causes

total 77

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FIG. 6. Regional treatment with Cytochalasin B (2.0/ig/ml for 10 min). (a) Shows the cytochalasin-containing agar in position against the early embryo.Note the position of the blastoderm, (b) Embryo

organogenic effects.Marthy (1972) was able to produce ab-

normalities of development with heat shockduring early cleavage stages. Since rela-tively low temperature heat shocks of shortduration disrupt normal mitosis and pro-duce such gross aberrations as internuclearbridges it is not surprising that normaldifferentiation in these embryos was impos-sible (Fig. 8).

From the above experiments it was con-cluded that there is a pattern of develop-mental information in the early embryoand that it apparently arises in the cortex atthe time of cytoplasmic streaming whichfollows fertilization and leads to the estab-lishment of the blastodisc. This hypothesisis similar to that put forth by Raven(seel961) and agrees with similar work ongastropods and scaphopods (Clement,1968; Cather, 1971; Verdonk, 1968; Wil-son, 1904 a, b; Cather and Verdonk, 1974;etc.).

EXPRESSION OF THE INFORMATIONAL PATTERN

The mechanism of expression of thecor-

shown in (a) after six days of development. Althoughthe treated area cellulated normally, organogenesiswas inhibited. X65.

deal information pattern is of interest. In aseries of tissue isolations and recombina-tions it has been possible to implicate theyolk syncytium as a primary organizeranalogous to the dorsal lip of the blastoporeof amphibians (Arnold, \965b). If thewhole eye primordium at stage 17 consist-ing of two cellular layers, the underlyingyolk syncytium, and the adherent yolk wasisolated in a culture medium, differentia-tion of the eye continued and an optic vesi-cle of normal appearance developed. If theouter two layers of cells were separatedfrom the yolk syncytium and isolated intonutrient media they would round up, sur-vive, but not differentiate. The cells sur-rounding the denuded yolk syncytiummigrate in to close the wound unless there isyolk leakage through the fragile yolk syn-cytium. If there is yolk leakage at the opera-tion site, wound closure is delayed and partof the yolk syncytium may be damaged. Incases where there was no yolk leakage,wound closure took place fairly rapidly(approximately three hours) and develop-ment continued. In these cases a completeembryo containing all the organs in their

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§4O

83

JM. i2 'A

STAGE OF DEVELOPMENT

6

5

3

2

I O w 12 14 15.

FIG. 7. Graphic representation of the effects ofCytochalasin B pulses (0.2 /xg/ml for 10 min) at varioustimes during development. The anomalies were as-signed arbitrary classes depending on severity and theembryos in each experiment were scored. Class one

normal positions was formed. If cells fromother embryos were dissociated by shakingand allowing reaggregation to occur, thereaggregated cells could be grafted onto

represents normal embryos and class six represents noorgan differentiation. The major sensitivity occurredduring the cyloplasmic streaming which formed theblastodisc. See text for details. (Redrawn from Arnoldand Williams-Arnold, 1974).

freshly denuded yolk syncytium. Thesereaggregates would attach to the surface,spread and eventually fuse with the woundclosing tissue. These grafted tissues can be

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EGG CORTEX OF THE SQUID •436

FIG. 8. Intranuclear bridges resulting from heattreatments similar to Manny's (1972) experiments(two focal levels of the same embryo). These Feulgen

dye labeled before grafting so they can becarefully followed on the host embryo.Development of the grafted tissues corre-lated with the position of the yolk syn-cytium onto which they were grafted. Thereciprocal operation was also performed. Ifthe outer cellular layers were removed,then a small portion of the yolk syncytiumcarefully excised, a small yolk leak would beproduced. When the migrating outer layersclosed the wound, the yolk would bepinched off from the embryo. These em-bryos developed organ deficiencies corre-lated with the position of the excised yolksyncytium. It was concluded from these ex-periments that a pattern of develop-mental information also exists in the yolksyncytium and it directs the cells that cometo lie over it to differentiate into the organsof that region. These results are in agree-ment with the type of inductive interactionreported by Clement (1968) and Cather(1967) for 1lyanassa.

Marthy (1973) attempted to refute theseresults on the basis of the inability to repeatthe results of the denuding experiments.

preparations show abnormalities of mitosis. Eventu-ally these bridges break with a loss of chromatin fromone or both nuclei. X90.

He incubated his embryos in sterile seawater to which no nutrients had beenadded and wound closure took considera-bly longer. Apparently, he did not attemptto graft dissociated-reaggregated cells ontothe yolk syncytium surface. Further, hewounded the embryo at the vegetal polewhich allowed extensive yolk leakage whichreduced the volume of the embryos thuschanging the spatial relationship of theembryonic surface. From his negative re-sults Marthy (1973) disagreed with thehypothesis that the yolk syncytium had anyinductive role in development and sug-gested that the yolk syncytium had onlya nutritive role in embryogenesis. By split-ting an early eye primordium, he claimed tohave produced reduplicated organs. How-ever, careful examination of his publishedphotographs suggests that the operationmerely tore the eye primordium into twosegments and his Figure 19 (1973) shows aneye with a disrupted retina, a partially sepa-rated lens, and a single iris. The posteriorportion of the lens is partially separatedinto two pieces but the anterior lens cap is a

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single structure. These results would beexpected if the anlage had simply been sur-gically bisected and later allowed to fuseback together.

Structural studies on the interrelation-ship of the blastoderm cells and the yolksyncytium can give partial insight into thepossible mechanism of communication andinformation transfer during organo-genesis. Recently, there has been consider-able interest in cellular junctions as possiblesources of intercellular communicationduring differentiation (Bennett, et al.,1972; Revel, 1974; Dixon and Cronly-Dillon, 1974; etc.). Furthermore, the pos-sibilities of electric communication betweendifferentiating cells has been discussed(Potter, et al., 1965; Loe wen stein, 1968; etc.)in reference to possible control of dif-ferentiation. What follows here is a pre-liminary study of the ultrastructure of theearly squid blastoderm which demonstratesthat structural connections exist whichwould allow communication via severalpathways between the cells of the blasto-derm and the blastoderm and the yolk syn-cytium.

Scanning electron micrographs of thepost-gastrulation blastoderm show manycellular interconnections (Fig. 9). The sur-face of the embryo is covered with a thinextracellular membrane which arises afterfertilization.The outermost cells of theblastoderm at stage 13 are fitted togetherlike paving stones and form a relativelysmooth outer surface. However, their innersurfaces more or less conform to the shapeof the adjoining cells. The lower cells of theblastoderm have irregular angular shapeswhich interlock with the surrounding cells.These lower cells lie in individual socket-like depressions of the yolk syncytium.

Scanning electron micrographs (Fig. 9)and transmission electron micrographs(Fig. 10 and 11) show many cytoplasmicprojections between the blastomere cellsand the blastoderm and the yolk syncytium.These projections vary greatly in length,diameter and direction but a "typical" cellhas an average of 42 such projections on itsviewable surface (Fig. 9, inset).The socket-like depressions of the yolk syncytium alsohave many cytoplasmic projections butthese tend to be mainly at the edges of the

hexagonally shaped depressions. Thereseems to be no apparent pattern of the pro-jections other than their tendency to occurat the corners of the cells. In thin sectionsthe projections may end with a gap junc-tion, in a small indentation, or freely in theintercellular space (Fig. 10a, and lOd; l la,1 lb, 1 ld).There are also many other typesof intercellular connections including gapjunctions (Fig. 10a), intercellular bridges(Fig. 10b), occluded intercellular bridges(Fig. 10c), and septate desmosomes (Fig.lOe). Frequently, the tip of the projection inan indentation is surrounded with vesiclesreminiscent of synaptic vesicles.

On the basis of structural evidence aloneit is not possible to establish that inter-cellular or transcellular communication isresponsible for the coordination ofdifferentiation but since such coordinationobviously occurs in all multicellular dif-ferentiating systems it is possible to specu-late that such communication might be re-lated to either the junctional complexes orthe cytoplasmic projections (see Sheridan,1974).

ORIGIN OF THE INFORMATIONAL PATTERN

Although the cytochalasin B experi-ments demonstrate that the informationalpattern in the egg cortex becomes or-ganized or activated at the time of blas-todisc formation, logic dictates that someprecursor pattern must be present beforethis time and this precursor pattern ulti-mately must arise in the oocyte during oo-genesis. The nature and organization ofthis precursor pattern is completely un-known but analogies with Raven's (1967)work on similar problems in gastropodsstrongly indicate that the follicularepithelium might be responsible for pro-gramming the oocyte. Therefore we havebegun to look at the ultrastructure of oo-genesis in Loligo. Despite the fact that thisapproach has obvious limitations andshortcomings, any insight gained at thispoint is helpful. What follows here is a briefsummary of work in progress and althoughit is painfully incomplete, it does suggestareas for further consideration. Recently,Bottke (1974) and Dhainaut and Richard

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FIG. 9. Scanning electron micrograph of the stage 13blastoderm. At this stage the whole blastoderm is ex-panding so the middle layer is becoming flattenedfrom a random multicellular layer to a sheet one cellthick. Many cytoplasmic bridges come from each cell

and connect to neighboring cells or to the yolk syn-cytium. x 1,150. The insets show details of the cyto-plasmic projections and the surface of the yolk syn-cytium. Upper, X3000; lower, x3,400.

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(1972) have studied oogenesis in two othercephalopods so a long general descriptionis unnecessary here. Instead, structurespossibly pertinent to the cortex or follicularepithelium-egg cortex interaction will bediscussed.

The ovary in small juvenile (ca. 22-35 mmmantle length) Loligo pealei ceases de-velopment in the fall and meiosis stops inProphase 1. Subsequent growth anddevelopment of the oocyte occurs while thenucleus remains tetraploid. With the onsetof spring, maturation of the oocyte pro-ceeds and development is asynchronous sothat in a given individual several stages ofmaturation can be found at one time. Cow-den (1968) divided the maturation of theoocyte in Loligo brevis into 10 stages and hisscheme will be used here with the additionof a prefollicular stage in which the follicu-lar epithelium has yet to be formed. Thevery early immature oocytes have one fol-licular cell associated with them. The nu-cleus occupies about 40% of the oocyte'svolume and has many large ring-shapednucleoli in it (Fig. 12). In stage III thesenucleoli apparently break down into ribo-somes and transverse the nuclear envelopewhere they formed a halo around the nu-cleus (Fig. 13). Later in development, theydisperse into the cytoplasm.

In stage III the follicular epithelium hascompletely surrounded the oocyte and theindividual cells have taken on a cubodialshape (Fig. 14). The plasma membrane ofthe follicular cells develops a number offolds on the surface in contact with the oo-cyte and these folds project into the oo-plasm for a short distance. This "fringeborder" excludes the organelles of the oo-cyte and appears to contain a uniformgranular layer of cytoplasm obviously dif-ferent than the rest of the ooplasm. Freezeetch preparations show that these mem-branes contain granular areas character-istic of gap junctions. The fringe border

gradually disappears and massive areas ofmicrovillar projections appear between theoocyte and the follicular epithelium (Fig.15). Large desmosome-like structures al-ternate with the microvillar areas (Fig. 15)and persist until formation of the chorionfinally separates the follicular epitheliumfrom the oocyte.

As the oocyte increases in size, the follicu-lar epithelium becomes highly folded andthe folds project into the oocyte so it be-comes extremely stellate. The plasmamembranes between the individual follicu-lar cells are retracted to the basal and apicalsurface so that the follicular epithelium be-comes a syncytium (Fig. 16). The mem-brane is apparently retracted by an activeprocess because either side of the mem-brane is paralleled by microtubules (Fig. 16,inset) and it becomes folded near the ex-terior or interior surface. Regional differ-ences do occur in the cytoplasm of this mas-sive syncytium; areas rich in endoplasmicreticulum or other cytoplasmic organellescan be found while other regions appearless specialized (Fig. 17).

Further development involves massiveyolk production, apparently by the Golgiapparatus (Bottke, 1974), until the cyto-plasm is stretched to a thin layer surround-ing the central yolk. This thin layer of cyto-plasm stains very heavily for RNA. Duringyolk accumulation, the follicular syncytiumfolds are withdrawn so that the mature oo-cytes have a smooth surface. Concomitantwith yolk synthesis, the chorion is formedby fusion of droplets formed in the endo-plasmic reticulum of the follicular syn-cytium.

At oocyte shedding the cytoplasm of theoocyte contains many irregular vesicles andis extremely basophilic (Fig. 18). There isno membrane apparent between the chor-ion and the oocyte surface. These irregularvesicles are not present after fertilizationand an extracellular membrane now covers

FIG. 10. Connections between cells of the blastoderm,(a) Gap junction, X33.000; (b) intercellular bridge,x 19,700; (c) occluded intercellular bridge, X53.000;(d) short process inserting into neighboring cells—notethe vesicles associated with the indentation,x 12,300;(e) septate desmosome between two surface cells,x 56,000; (f) process from one cell contacting twoother cells, one by a gap junction, x 19,800.

FIG. 11. Connection between the blastoderm cells andthe yolk syncytium. (a) Short projections with vesiclesassociated with the indentation. x52,000. (b) Longprojection underlying a neighboring cell X 16,400. (c)Gap junction and projection from the yolk syncytiumx 12,800. (d) Long cellular process penetrating deepinto the yolk syncytium. Note the vesicles at the base ofthe indentation, x 27,700.

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EGG CORTEX OF THE SQUID 4+1

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442 JOHN M. ARNOLD AND LOIS D. WILUAMS-ARNOLD

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FIG. 18. Mature oocyte surface just prior to shedding.Note the granular density of the cytoplasm and thenumerous transparent vesicles (v). Irregular microvilli

(m) cover the surface and lie against the chorion (ch).The yolk platelets (yp) are membrane bounded,x 15,300.

the entire zygote surface suggesting a corti-cal reaction has occurred to form a fertiliza-tion membrane.

From the above observations, it wouldappear that the surface of the developingoocyte has specializations which change

during its developmental history. First the"fringe border" of the follicular epitheliumappears and excludes organelles from thesurface of the oocyte and a specializedcytoplasm is present between the mem-branous folds. The presence of gap junc-

FIG. 12. Primary oocyte with one follicular cell (fc).There are many complex nucleoli at this stage. Laterthe follicular cells will undergo mitosis and eventuallycompletely surround the developing oocyte. x2,800.

FIG. 13. Movement of nucleolar material across thenuclear envelope following breakdown of the nucleoli.Arrows indicate RNA positive granules. X33.500.

FIG. 14. Stage III follicular epithelium cells. Theplasma membrane of the follicle cells has formed foldswhich project into the ooplasm. The cytoplasm be-tween these folds appears denser than the rest of theooplasm and organelles are excluded from this areax 10,800. The inset is a freeze fracture preparation ofone of the folds showing the granules characteristic ofgap junctions, x 200,000.

FIG. 15. Interface of oocyte and follicular epitheliumafter disappearance of the "fringe border" shown inFigure 14. Note the extensive areas of microvilli (m)and the desmosome-like connection (d). The Golgiapparatus appears to be synthesizing yolk (arrow),x 29,000.

FIG. 16. Transition of the follicular epithelium fromindividual cells to a syncytium. Note the membranesappear to be withdrawn (arrows) x 2,400.The insetshows microtubules associated with the retractingmembrane X67.500.

FIG. 17. Regional differences between two areas of thefollicular syncytium. Note the apical position of themitochondria on one side (arrows) and the differencesin organelle distribution, x 2,500.

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tions in these membranes suggest com-munication between the early oocyte andthe follicular cells. Later these membran-ous folds disappear and areas of microvilliappear between an extensive network ofdesmosomes. During this time the oocyte isincreasing tremendously in size and it isobvious that precursor materials must beactively passed from the follicular cells tothe oocyte although no special pathways forlarge materials, such as particles or vesicles,are evident. As the yolk is synthesized, itforces the cytoplasm to form a thin surfacelayer which may possibly still contain all thenucleolar RNA which was passed into thecytoplasm early in development. Althoughit is not possible to distinguish any specialcytoplasmic accumulations correlated withembryonic development, the regional dif-ferences in the follicular syncytium are al-most surely reflected in the adjoining oo-cyte surface. This work is obviously pre-liminary and hard conclusions cannot yetbe drawn, but it suggests that the egg cortexhas a developmental history complexenough for it to develop a latent pattern ofinformation which could later function indifferentiation. Exactly where this patternresides, either the cytoplasm or plasmamembrane, is a question still to be attacked.

CONCLUSIONS

From the above experiments and obser-vations, it is possible to conclude that thereis a pattern of developmental informationresident in the egg cortex which becomesrather rigidly fixed early in development.Possibly, this fixation of fate arises at thetime of, and is correlated with, the cyto-plasmic streaming which forms the blasto-disc. This organogenetic pattern is eventu-ally imposed upon the blastoderm cells thatcome to cover organ-specific regions of theyolk syncytium via cellular interactionssimilar to those which occur in other mol-luscs (Cather, 1971; Clement, 1968, 1976).These results are in close agreement withthe conclusions of Raven (1961) onLymnaeain which it was possible to demonstrate thata pattern of cortical information existed inthe egg cortex. Verdonk (1968) demon-strated a similar pattern in the cortical re-

gion of Dentalium.The exact nature of thecortical information is still open to specula-tion but the work of Dohmen and Verdonk(1974) in which they demonstrated an RNAbody present in the polar lobe of Bithyniaand the work of Newrock and Raff (1975)showing polar lobe control of proteinsynthesis via preformed raRNA would leadus to speculate that the informational pat-tern is specific RNA messages. The fact thatthere are high levels of RNA present in thecortex of the mature egg would strengthenthat speculation. Possibly the syncytial fol-licular epithelium provides the necessarypattern to the RNA during oogenesis eitherdirectly by transfer of messenger across theoocyte membrane or indirectly by selec-tively programming the messenger formedin the oocyte proper. This messenger thendirectly or indirectly controls selective acti-vation of parts of the genome duringorganogenesis. The connections whichexist between the cells of the blastodermand the yolk syncytium could provide astructural basis for the developmentalcommunication which undoubtedly mustoccur during differentiation.

It would seem that the cephalopod em-bryo represents the logical extension of themechanisms of differentiation found inother molluscs. There is developmentalinformation necessary for normal de-velopment in the polar lobe and D quad-rant. This information is passed to themicromeres whose cells differentiate alongspecific pathways dependent upon wherethey contact specific parts of the macro-meres (see Cather, 1971). As the cephalo-pods evolved, the developmental informa-tion resident in the egg became highly re-gionalized in the surface of the large yolkedegg. The blastoderm cells utilize this pat-tern of information and differentiate in ac-cordance with their position. Once theyhave received a set of developmental in-structions (i.e., their genome is activated)the changes are permanent and irreversibleand differentiation occurs along specificpathways without modification. In otherterms, the egg cortex-yolk syncytium com-plex is a morphogenetic inductive mapsimilar to that found in the primary or-ganizer of vertebrate morphogenesis

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where similar cortical patterns and cellularinteractions (inductions) are well known.

NOTE ADDED IN PROOF

Since this paper was written in the springof 1975, additional data have become avail-able concerning the changes at the egg sur-face following fertilization. Electron mi-croscopy has shown that the extensive cyto-plasmic vesicles appearing in Fig. 18 slowlymigrate to the surface following fertiliza-tion where they fuse and become incorpo-rated into the plasma membrane of the zy-gote. Since cytochalasin B is known to blocksimilar exocytosis in other organisms(Copeland, 1973) the effect of regionallyapplied cytochalasin B on organogenesismay be due to a failure of the incorporationof the cytoplasmic vesicle membrane intothe zygote surface membrane. This wouldimply that the informational pattern is in-corporated in the plasma membrane andthat "activation" or "release" of the de-velopmental information is related to struc-tural changes in the plasma membrane andthat the membrane itself is informational.De Terra (1975) has demonstrated corticalcontrol of DNA synthesis in Stentor. If this isthe case, the role of cytoplasmic streamingin the release of the informational patternmay be of secondary importance. Furthersupporting data will be published else-where.

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The Egg Cortex Problem as Seen Through the Squid Eye

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