/ . Embryol. exp. Morph. Vol. 31, 2, pp. 423-433,1974 4 2 3

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The structure of a morphogeneticcytoplasm, present in the polar lobe of Bithynia

tentaculata (Gastropoda, Prosobranchia)

By M. R. DOHMEN1 AND N. H. VERDONK1

From the Zoological Laboratory, University of Utrecht, The Netherlands

SUMMARYIn the first polar lobe of the egg of Bithynia tentaculata a cup-shaped mass of small vesicles

is described, which fills the greater part of the lobe. It is named the 'vegetal body'. Withmethyl green-pyronin the vegetal body stains clearly, but after treatment with RNase nostaining occurs, thus indicating the presence of RNA. The first polar lobe of Bithynia is ofgreat importance for further development of the embryo and it is argued that the vegetal bodycould be a morphogenetic cytoplasm, responsible for the developmental effects of the polarlobe.

INTRODUCTION

In the eggs of many annelids and molluscs the first cleavages are characterizedby the appearance of polar lobes. The contents of these lobes are of great im-portance for further development, as is shown by experiments involving removalof the lobe or deletion of cells receiving the lobe contents. In Ilyanassa, for in-stance, lobe-dependent structures are: foot, eyes, operculum, statocysts, shell,heart and intestine (Crampton, 1896; Clement, 1952, 1956, 1962; Cather, 1967;Atkinson, 1971). The successively appearing lobes do not all have the sameeffect. In Dentalium (Wilson, 1904), Sabellaria (Hatt, 1932; NovikofT, 1938a, b)and Mytilus (Rattenbury & Berg, 1954) development of the apical tuft of thelarva is dependent on the presence of the first polar lobe, but not on the secondone.

Attempts to identify polar lobe factors have given very poor results up till now.In many cases differences in composition can be demonstrated between thecytoplasm of the polar lobe and the rest of the egg (Pitotti, 1947; Clement &Lehmann, 1956; Pasteels & Mulnard, 1957; Reverberi, 1958, 1970; Berg &Kato, 1959; Collier, 1960a, b; Dalcq & Pasteels, 1963; Crowell, 1964). Butcentrifugation experiments (Clement, 1968; Verdonk, 1968) have establishedthat in Ilyanassa and Dentalium the morphogenetic factors are not located in thedisplaceable components of the polar lobe cytoplasm. Unfortunately ultra-structural and histochemical studies on displacement of cytoplasmic componentsin polar lobes of centrifuged eggs are lacking.

1 Authors'1 address: Zoology Laboratory, Janskerkhof 3, Utrecht, The Netherlands.27 E MB 31

424 M. R. DOHMEN AND N. H. VERDONK

Accumulation of morphogenetic substances in special areas of the egg andsegregation of these accumulations into special blastomeres are not restricted topolar-lobe-forming organisms, but can be demonstrated in many other animals.In spite of such a widespread occurrence of the segregation phenomenon,identification of the morphogenetic substances in the segregated cytoplasm hasnot been successful. Even in the polar granules of insect eggs, 'the only caseknown where developmentally significant information is localized in organellesthat can be seen and followed during development' (Mahowald, 1971), it is notknown whether it is the protein or the RNA component which is developmentallysignificant. We describe a structure in the polar lobe of Bithynia tentaculatawhich could be a second case of an organelle storing morphogenetic substances.

METHODS

Bithynia tentaculata is a freshwater prosobranch snail which can be collectedin ditches around Utrecht, Holland. In aquaria in the laboratory, egg masses aresoon deposited on plant leaves. These egg masses consist of capsules whichcannot be separated from each other. The tough capsule membrane is firstperforated with a very sharp knife in order not to compress the egg. Then the eggcan be removed from the capsule with a hair-loop and the viscous capsule fluidcan be washed off in tap-water.

Fixation and staining for light microscopy. Eggs fixed in Zenker's fluid werestained with Heidenhain's iron haematoxylin and eosin for general study or withmethyl green-pyronin for nucleic acids (Brachet, 1942,1953). For the latter staineggs usually were fixed in ethanol-acetic acid (3:1) for a more vivid stain. For theFeulgen method, eggs were fixed in ethanol-acetic acid (3:1) and either stained assections or in toto according to the method of van den Biggelaar (1971).

Fixation and staining for electron microscopy. Eggs were fixed for 3 h at 4 °C ina mixture of equal parts 2 % glutaraldehyde and 2 % osmium tetroxide, both in0-1 M Na-cacodylate buffer at ph 7-4. The eggs were then washed in buffer,oriented in agar, dehydrated in a graded series of ethanol, followed by propyleneoxide, and embedded in Epon 812. Sections were stained for 10 min in a saturatedsolution of uranyl-acetate in 70 % methanol, followed by 1 min in a lead solutionaccording to Reynolds (1963).

RESULTS

I. Observations with the light microscope

At the vegetal pole of freshly laid eggs of Bithynia, in which the germinalvesicle is still present, a densely staining cytoplasm is present. At this stage thedense plasm is intimately connected with the surface of the egg (Fig. 1). After thegerminal vesicle has disappeared, the dense cytoplasm rises from the surface(Fig. 2) and soon assumes the shape of a cup with the open side towards thevegetal pole. Because of its position we call it the 'vegetal body'. It stays at the

The polar lobe o/Bithynia 425

• • • k *

.

Figs. 1-5. Light micrographs of eggs of Bithynia tentaculata stained with ironhaematoxylin and eosin. x 350.Fig. 1. Egg just after oviposition with the germinal vesicle still present. Arrowindicates dense cytoplasm at the vegetal pole.Fig. 2. Egg after breakdown of the germinal vesicle. Dense cytoplasm (arrow) freefrom the surface.Fig. 3. Egg at first cleavage. Arrow indicates dense cytoplasm now present as cup-shaped vegetal body in the polar lobe.Fig. 4. Two cell stage with the vegetal body (arrow) at the vegetal side of the CD-blastomere.Fig. 5. Section through the CD-blastomere at the beginning of second cleavage.Arrow indicates the second polar lobe, filled with clear cytoplasm but missing thevegetal body.Fig. 6. Light micrograph of an egg at first cleavage, stained with methyl green-pyronin. The vegetal body (arrow) is densely stained.

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426 M. R. DOHMEN AND N. H. VERDONK

Fig. 7. Electron micrograph of the first polar lobe with vegetal body. Arrow points toa dense body. AZ, Attachment zone; L, lipid; M, mitochondrion, x 9700.

vegetal pole, surrounded by clear cytoplasm, until first cleavage, when a polarlobe is formed. Compared with other polar lobes, described in molluscs, thislobe is remarkable in several respects: it is extremely small (diameter 20-30 /an)and it is usually nearly free from yolk granules.

The polar lobe of Bithynia 427The vegetal body is incorporated in the first polar lobe (Fig. 3). After first

cleavage the vegetal body is transferred with the lobe to the CD-blastomere.During the whole 2-cell stage it remains at the vegetal side of the CD-blastomere(Fig. 4). At second cleavage the vegetal body suddenly disappears at about thestart of anaphase. A second polar lobe is formed at this stage, which, however, isnever as completely separated as the first polar lobe and remains broadly con-nected with the CD-blastomere. The second lobe contains a clear cytoplasm butthe vegetal body is no longer present (Fig. 5). In order to study the chemicalnature of the vegetal body, we stained it with the Feulgen method for the presenceof DNA. Both in sections and in whole mounts the staining was negative. Withmethyl green-pyronin the body stains clearly (Fig. 6). After pre-treatment of thesections with RNase (Worthington Biochem. Corp.) no staining of the vegetalbody occurs, indicating the presence of RNA.

II. Observations with the electron microscope

The vegetal body is a cup-shaped mass of small vesicles and among them someribosomes, mitochondria, large vesicles and dense bodies can be found (Fig. 7).The vesicles have a diameter of 50-100 nm and most of them are filled with adark-staining substance (Fig. 8), probably RNA, as RNase treatment of thicksections makes the vegetal body almost completely disappear. The vesicles arenot homogeneously distributed, but they are arranged in an irregular network,with vesicle-free areas in between (Fig. 7). The same type of vesicles also forms athin layer under the membrane of the stalk which connects the polar lobe to theegg and this layer extends to a limited distance (about 15 /im) under the neigh-bouring egg-membrane (Fig. 9). Occasionally a small cluster of vesicles can befound outside the polar lobe in the egg cytoplasm, close to the stalk. In view oftheir distribution it seems possible that a few vesicles will come to lie in the AB-blastomere at first cleavage.

Preliminary centrifugation experiments indicate that the vegetal body is hardto displace. Even when part of the uncleaved egg is centrifuged off, the vegetalbody stays in most cases in its original place and when the egg is left to cleave aftercentrifugation, the vegetal body is found in the polar lobe at first cleavage. Theseresults indicate that the vegetal body is firmly attached to the vegetal pole andthat there is a strong cohesion of the body itself. Still, no special devices respons-ible for attachment and cohesion can be found. As attachment is likely to takeplace by means of the cytoplasmic zone lying between the vegetal body and thevegetal pole, we called this zone the 'attachment zone' (Fig. 7).

In the polar lobe there is also to be found a concentration of multivesicularbodies. They often contain densely coated vesicles, which can easily be dis-tinguished from the vesicles making up the vegetal body. The multivesicularbodies are often ruptured and their coated vesicles may be seen lying in strings inthe attachment zone (Fig. 10).

M. R. DOHMEN AND N. H. VERDONK

Fig. 8. Detail of the vegetal body, showing the small vesicles of which it consists.Most vesicles are completely or partly filled with a dark-staining substance, probablyRNA. x 105000.

Fig. 9. Superficial layer of the stalk (ST) of the polar lobe and its implantationregion on the egg, showing a layer of small vesicles under the membrane, x 27 700.

The polar lobe o/Bithynia 429

Fig. 10. Multivesicular bodies {MVB) in the polar lobe. One of them is ruptured(double arrow), releasing coated vesicles (arrow) into the attachment zone {AZ).x 27 700.

430 M. R. DOHMEN AND N. H. VERDONK

DISCUSSION

The facts that are known at present about the vegetal body are all in agreementwith the view that the vegetal body could contain the morphogenetic factorsinvolved in determining the lobe-dependent structures.

In Bithynia the significance of the polar lobe for development has been estab-lished by removing the lobe at first cleavage. No adult organs like eyes, tentacles,shell, etc., develop in lobeless embryos (Verdonk & Cather, 1973). The vegetalbody fills the greater part of this polar lobe so it is obvious to assume that thedevelopmental information is located in the vegetal body and most probablyinside the vesicles. After first cleavage the vegetal body is transferred with thepolar lobe to the CD-blastomere. In isolation experiments (Verdonk & Cather,1973) the AB-blastomere forms hardly any adult structures, whereas the CD-blastomere, containing the polar lobe, forms most adult structures. At secondcleavage the vegetal body disappears rather suddenly between meta- and ana-phase, some time after the disappearance of the nuclear membrane. Aftersecond cleavage the C- and D-blastomeres have the same morphogenetic capaci-ties (Verdonk & Cather, 1973). As the vegetal body disappears before thecleavage furrow separates the C- and D-blastomeres, the substances containedin the vegetal body can spread over both cells and in this way give them the samedevelopmental potentialities.

Other arguments in favour of a morphogenetic role are the fact that thevesicles forming the vegetal body are typical storage vesicles, which could keepthe morphogenetic factors inactive until their time has come to act, and the factthat the vegetal body contains RNA, which is an obvious candidate for the roleof morphogenetic factor.

A large structure such as the vegetal body of Bithynia cannot easily be over-looked, still nothing like it has been found in thoroughly investigated species likeIlyanassa (Pucci-Minafra, Minafra & Collier, 1969; Gerin, 1972), Mytilus(Humphreys, 1964) and Dentalium (Reverberi, 1970). This probably means thatthe vegetal body is an exceptional structure, but the vesicles composing it couldwell be a general carrier of developmental information. If in other animals thesevesicles should not be as numerous as in Bithynia and scattered over a largerarea, they could probably escape attention.

In all species investigated thus far, several larval or adult structures are de-pendent on one lobe, so it is to be expected that this lobe contains several kindsof morphogenetic substances which become separated during cleavage until atlast each one reaches its own target cell. Such a mechanism might be deducedfrom experiments with Dentalium. Here the apical tuft and the post-trochalregion are both dependent on the first polar lobe. If 60 % of the lobe is removed,however, larvae develop with an apical tuft and a reduced post-trochal region(Geilenkirchen, Verdonk & Timmermans, 1970). This result points to thepossibility of at least two different factors: an apical tuft factor located in the

The polar lobe of Bithynia 431animal region of the lobe, which is not removed, and a post-trochal region factorin the vegetal region. The finding in Bithynia of a mass of apparently identicalvesicles, whose contents probably determine several adult organs, points toanother possible mechanism: a polar lobe might contain one kind of morpho-genetic factor only, which in some way, maybe by unequal distribution over thecells, determines the development of several organs.

The lack of any visible mechanical structure holding together the vegetal bodyand attaching it to the vegetal pole is another problem. Special cytoplasmsaccumulated under a particular area of the cell periphery are a common featurein many eggs, but they do not seem to react in the same way to centrifugal force.The vegetal body of Bithynia and the polar lobe factors of Ilyanassa and Dentaliumare not displaced by centrifugation, while the animal pole plasm and the sub-cortical accumulations of Lymnaea, for instance, are easily disrupted and dis-persed (Raven, 1945; Raven & Brunnekreeft, 1951; Raven & van der Wai, 1964;Raven, 1967). In all cases there is probably some kind of attraction exerted byparticular areas of the egg periphery upon certain cytoplasmic components, butwe can only speculate about their nature.

The last problem to be discussed is the concentration of multivesicular bodiesin the polar lobe of Bithynia. Very little is known about multivesicular bodies.In eggs they seem to be formed by transformation of yolk granules (Pasteels & deHarven, 1963) and they contain a high concentration of phosphatases (Dalcq,1962; Dalcq & Pasteels, 1963). Reverberi noted multivesicular bodies in thepolar lobe of Dentalium, and the polar lobe of Sabellaria shows a high concentra-tion of phosphatase (Dalcq & Pasteels, 1963), which could also result from a highconcentration of multivesicular bodies. No multivesicular bodies have beenreported in the polar lobes of Ilyanassa and Mytilus. Too little is known as yet toenable us to suggest a function for the multivesicular bodies in polar lobes.

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(Received 5 July 19'/73)