A morphometric comparison of dissimilar early development in sibling ...

Roux's Arch Dev Biol (1992) 201:243-256

Roux'sArchivesof

velopmental

Biology

© Springer-Verlag 1992

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida, Polychaeta) Stephan Schneider, Albrecht Fischer, and Adriaan W.C. Dorresteijn Zoologisches Institut (Abteilung1) der Universit~tMainz, Saarstral3e25, W-6500 Mainz, Federal Republic of Germany Received March 10 / Accepted March 13, 1992

Summary. Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to ."A.W.C. Dorresteijn

Key words: Polychaete - Segregation - Spiral cleavage - Phylogeny Cell cycle

Introduction "The cleavage of the ovum takes place with a precision and regularity which oft-repeated examination only renders more striking and wonderful" (E.B. Wilson, The cell-lineage of Nereis. A contribution to the cytogeny of the annelid body. J Morphol 6, 1892 p. 377)1. In spiralian embryos, cleavages divide the egg in an invariant and species-specific manner into blastomeres which differ in size. The blastomeres can be recognized by their proportions and by their position in a stage- and speciesspecific cell pattern. Each blastomere is endowed with a predictable developmental fate. These facts have long been established (review, Schleip 1929). Using modern methodology (video recordings of living embryos, improved histological methods and computer-aided morphometry for fixed material), one of us has recently supplemented these classical observations with cytological parameters such as cell, nuclear and yolk volumes, and cell cycle duration for individual blastomeres in the embryo of the polychaete Platynereis dumerilii (Dorresteijn 1990). Blastomeres in early spiralian embryos diversify in many respects, and ever since blastomere diversification and its linkage to cell fate was first detected, it has been suggested to reflect early determination of embryonic cells. Indeed, the fate of blastomeres in the embryos of P. dumerilii (Dorresteijn et al. 1987; Dorresteijn and Eich 1991) and many other spiralians may be altered if the cleavage pattern is altered experimentally. It has thus been concluded that at least part of the visible diver1 The authors dedicate this paper to the memory of E.B. Wilson (1856-1939), whose pioneering paper on the early development of Nereis appeared 100 years ago. It still is a source for careful observation and of astonishinglyclear-sightedstatements and postulates on the nature of developmental decisions, some of which Wilson verifiedhimselfin later years.

244 sification a m o n g b l a s t o m e r e s is a p r e r e q u i s i t e for n o r m a l development. T h e i m p o r t a n c e o f these d e v e l o p m e n t a l p a r a m e t e r s for m o r p h o g e n e s i s m a y also be e v a l u a t e d b y e x a m i n i n g t h e m in e m b r y o s w h i c h are m o r p h o l o g i c a l l y distinct, b u t w h i c h are closely r e l a t e d t a x o n o m i c a l l y . Specimens o f Platynereis massiliensis c a n n o t be d i s t i n g u i s h e d f r o m sexually i m m a t u r e P. dumerilii, a s y m p a t r i c sibling species. Nevertheless, the early d e v e l o p m e n t o f P. massiliensis ( f o r m e r l y called a " n e r e i d o g e n i c f o r m " o f "Nereis dumerilii"), as d e s c r i b e d by W i s t i n g h a u s e n (1891), a p p e a r e d to differ r e m a r k a b l y f r o m t h a t o f P. dumerilii, as d e s c r i b e d b y D o r r e s t e i j n (1990). E m b r y o s o f P. massiliensis seemed to d i s p l a y a different b l a s t o m e r e p a t t e r n , to d e v e l o p m u c h m o r e slowly a n d to skip the t r o c h o p h o r e stage w h i c h is w e l l - d e v e l o p e d in P. dumerilii. In this p a p e r , we wish to describe the cleavage o f P. massiliensis at the s a m e level o f detail as we h a v e r e c o r d e d t h a t o f P. dumerilii a n d to c o m p a r e b o t h courses o f d e v e l o p m e n t . By this c o m p a r i s o n we wish to d e t e r m i n e which o f the p a r a m e t e r s o f early d e v e l o p m e n t r e m a i n e d u n a f f e c t e d b y the p r o c e s s o f s p e c i a t i o n a n d w h i c h are o p e n for v a r i a t i o n even a m o n g closely r e l a t e d species. Since the result o f m o r p h o g e n e s i s l o o k s identical in the two species, those cleavage p a r a m e t e r s which are c o n s e r v e d r a t h e r t h a n those which v a r y can be r e g a r d e d as p a r t i c u l a r l y i m p o r t a n t for m o r p h o g e n e sis. Sibling species with identical a d u l t m o r p h o l o g y b u t different r e p r o d u c t i v e b i o l o g y , g a m e t e m o r p h o l o g y a n d e m b r y o g e n e s i s are f r e q u e n t a m o n g p o l y c h a e t e s ( D u r c h o n 1955). P. dumerilii a n d P. massiliensis were recognized as distinct species b y H a u e n s c h i l d (1951), a n d m a n y cryptic p o l y c h a e t e species o b v i o u s l y r e m a i n to be d e t e c t e d as s h o w n b y the recent r e p o r t s o f W e i n b e r g et al. (1990) o n Nereis "acuminata "' a n d o f S a t o a n d T s u c h i y a (1991) o n Neanthes "japonica". A l t e r n a t i v e sets o f r e p r o d u c t i v e a n d d e v e l o p m e n t a l traits seem possible for a n d realized in m a n y p a i r e d species o f nereids a n d o t h e r p o l y c h a e t e s . Here, we wish to c o m p a r e s o m e a l t e r n a t i v e traits o f r e p r o d u c t i v e b i o l o g y in P. massiliensis a n d P. dumerilii a n d discuss their i m p a c t on d e v e l o p m e n t a n d speciation.

Materials and methods Animals and reproductive biology. Platynereis massiliensis were collected on the Mediterranean coast at Banyuls-sur-Mer (France). Groups of tubes containing adults or brood can be collected from the basal part of young thalli of the intertidal green alga, Ulva lactuca. The animals were carefully removed from their tubes and put into flat plastic containers with seawater. In the laboratory, they were kept at 18° C with an artificial light regime (16 h light: 8 h dark). The worms were fed spinach, algae and fish food (Tetrarain) as described for P. dumerilii by Hauenschild and Fischer (1969). Since P. massiliensis does not metamorphose and spawns within the tube, the tubes had to be regularly inspected for the presence of eggs or developing embryos. Eggs are deposited a few hours before dusk. The moment and mode of fertilization still remain unknown. In his description of this species in 1951, Hauenschild

raises the possibility of self-fertilization. Indeed, we have occasionally found cleavage stages within the coelom of the female. The role of the male phase partner is thus unsettled; it may only serve for brood care.

Fixation and morphometry. A single brood of P. massiliensis may vary from 80-240 embryos. To collect morphometric data on the size and composition of blastomeres during early embryogenesis, small aliquots (5-7) of eggs were fixed every hour. After fixation for 2-3 h in a mixture after Flemming (Romeis 1989), containing 15 ml 1% chromic acid, 4ml 2% OsO~ and I ml acetic acid, the embryos were rinsed in tap water several times, oriented in 2% agar, dehydrated in graded ethanols and embedded in Araldite. After sectioning (1 lam) and staining with methylene blue/Azure II (Richardson et al. 1960), every fifth section was drawn on transparent paper and was digitized using a GENIUS graphic tablet and an 80286 personal computer with an 80287 mathematical coprocessor (Software: Autocad V 9.0; Autodesk, Basel, Switzerland). Morphometric data are given as percentages of total egg volume unless stated otherwise. Cleavage chronology. In contrast to the observations made on the development of P. dumerilii, in which development is rapid and the embryos are favourable objects for photography and videorecording, the opaque embryos of the sibling species P. massiliensis develop slowly. For recording, 2-3 embryos were placed in a drop of seawater and surrounded by a small ring of cardboard (400 gm thick) soaked with liquid paraffin and covered by a cover slip. Although this allowed several hours of development, the preparation eventually had to be replaced by a new set of eggs of the same batch. The progress of development was recorded on U-matic tape with a CR 6650 TL time lapse recorder (JVC, Friedberg, FRG) and photographed on A G F A Pan 25 ASA. Sequential fragments were then combined with the observations of cleavages after DNA staining with fluorescent markers (see below) to create the cell lineage diagram (Fig. 3) covering the first 21 h of development. Fluorescence microscopy. The embryos of P. massiliensis are extremely opaque and do not allow observation of nuclear breakdown, the event marking the end of a cell cycle. In order to study cleavage asynchronies, the nuclear DNA was stained by fluorescent markers. During the S-phase of an early cleavage cycle, some of the embryos were incubated in bromodeoxyuridine (BrdU: 5 mM BrdU and I mM fluordeoxyuridine in seawater; Gratzner 1982, modified by Plickert and Kroiher 1988) and fixed at regular intervals in methanol at - 2 0 ° C. After a double rinse in 0.1 ~ Na2HPO4, the embryos were incubated in 0.4 M glycine for 1-2 h. Denaturation was accomplished by treatment in 2 N HC1 for 35 min. After a short rinse in PBT [phosphate-buffered saline (PBS) +0.5% Triton XI00 +0.2% bovine serum albumin (BSA)], embryos were treated with 0.01% (w/v) Protease K (Sigma Deisenhofen, FRG) in seawater at 37° C for 15 min. After a l h rinse in PBT, the embryos were incubated in 100-200 gl of the primary antibody, mouse-anti-BrdU-IgG (Amersham Braunschweig, FRG) over night. After thorough rinsing in PBS, the secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse, was applied for 3 h. Superfluous fluorescent stain was washed out by rinsing in PBS, and finally the embryos were put into glycerol. Small aliquots of embryos were introduced into a drop of DABCOglycerol [25 mg DABCO (Sigma; 1,4-diazabicyclo-2,2,2-octan) in I ml PBS added to 10 ml of glycerol] to reduce bleaching. The preparations were studied with an epifluorescence microscope (Axioskop; ZEISS, Oberkochen, FRG) using the FITC excitation filter combination. In the living embryos, chromatin was rapidly stained after introducing them into a solution of 10 ~g bisbenzimid (Hoechst 33342; Sigma)/ml seawater. After transfer to seawater, living embryos could be studied under an epifluorescence microscope (see above) using 380 nm excitation wavelength. In all cases, photomicrographs were taken on AGFA Pan 400 ASA film using an Olympus OM4 Ti camera.

245

Fig. l. Unfertilizedegg of Platynereis massiliensis surrounded by the vitelline envelope ( I0 and a thin layer of egg jelly (J). The oocyte nucleus (dark spot) is marked by an arrowhead. The longitudinalaxis represents the future dorsoventral axis. Scale bat', 100 gm Fig. 2. Living two-cell stage of P. massiliensis, viewed from the animal pole. The smaller AB-blastomere (below) marks the future ventral side of the embryo. Scale bar, 100 gm

Results

Early development of P. massiliensis has been analysed from polar body formation onwards, with a complete cell lineage up to the 62-cell stage and additional observations on the development of the larvae and the 3segmented young worm. Fertilization was never observed in this species, and artificial fertilization proved unsuccessful in our hands. Therefore, metaphase of the first cleavage division was taken as the time of reference for working out a cleavage schedule. Due to the long course of cleavage in this species, we could record only fragments of the whole cleavage period for single embryos by time-lapse video recording. Additionally, we have determined the duration of interphases by using fluorescently-stained mitotic nuclei in both living and fixed embryos (Fig. 7). Cell volumes and the volumes of different components of the blastomeres (yolk and lipid versus clear cytoplasm) were determined from sections of fixed embryos by computer-aided morphometry.

Egg architecture and development prior to cleavage

The full-grown egg of P. massiliensis is ellipsoid (Fig. 1). The shortest diameter (250-290 gm) defines the animalvegetal axis of the egg (see below). An equatorial section of the egg is elliptic, with shorter and longer diameters of 260-300 gm and 290 350 lain, respectively. Size variation is greater in the eggs of P. massiliensis (_+8% in volume; n = 7 ) than in those of P. dumerilii (_+5.6%; n = 14), and unfertilized eggs of the former species dis-

play a triradial symmetry, whereas those of the latter show only a single axis of rotation (Dorresteijn and Kluge 1990). The volume of the P. massiliensis egg is much greater (approximately 1.6 x 107 gm 3) than that of the P. dumerilii egg (approximately 1.4 x 106 gin3), due to its much higher yolk content. We include both protein yolk bodies and lipid droplets under the term yolk in this paper. In P. massiliensis, yolk accounts for 86-93% of the egg volume, but in P. dumeriIii zygotes only for 64%. The absolute amount of "clear cytoplasm", i.e. cytoplasm free of yolk, on the other hand is only four times that of P. dumerilii. Whereas the egg of P. dumerilii is extremely rich in cortical granules and forms a relatively thick (300 gin) egg jelly coat after fertilization by extrusion of these granules, the egg of P. massiliensis contains relatively few granules and, in comparison, forms a moderate jelly layer (50-70 gm thick; see Fig. 1) after fertilization. In fertilized eggs, the animal pole becomes obvious as a yolk-free area, 40 gm in diameter. Above this area, the first and second polar bodies appear. Shortly after meiosis, the yolk-free cytoplasm forms an axial plug extending about 200 ~tm from the animal pole into the centre of the zygote. The clear cytoplasm then wells up towards the surface forming a yolk-free spot which reaches a diameter of 200 gm before first cleavage. Concomitantly, yolk and lipid migrate towards the vegetal pole and become compacted in the course of the subsequent cleavages. This process of ooplasmic segregation is similar to that observed in the zygote of P. dumerilii except that it takes 240 min from formation of the first

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p o l a r b o d y till first cleavage in P. massiliensis and, at the same t e m p e r a t u r e (18 ° C), only a b o u t 40 rain in P. dumerilii. Early d e v e l o p m e n t in general is m u c h slower in P. massiliensis as s h o w n b y c o m p a r i s o n of the cleavage schedule of this species with t h a t of P. dumerilii (Fig. 3).

Fig. 3. Cell lineage tree and time course of early embryogenesis at 18° C in the sibling species Platynereis massiliensis (heavy lines) and, for comparison, P. dumerilii (dotted lines; redrawn from Dorresteijn 1990), The time scale for P. dumerilii (below) has been standardized as to fit the cell lineage tree of both species. Standardization was based on determining, for both species, the mean of all intervals between first and each of the following cleavages included in the diagram. Mean developmental time of P. massiliensis to reach a certain cell stage takes 3.7 times longer than for the same cell stage in P. dumerilii. As indicated by the time scale above, we were not able to time the moment of fertilization in P. massiliensis, but the time course covers 21 h of early development (0 h is defined by metaphase of first cleavage). The data of this lineage are combined data from video-time-lapse recordings and observations of fluorescent DNA stainings in living and fixed embryos. The asymmetries in the sequence of divisions among both species are similar in all cell lines. However, some cells of P. mass# liensis seem "accelerated" with respect to identical cells of the other species. Especially, the cells 2a 1, 2b 1, 2c 1, 3A, 3B, 3C and all cells of the 2d cell line proliferate at a more rapid pace than do those of the P. dumerilii embryo. P.B., polar body formation

First cleavage As in P. dumerilii, first cleavage is m e r i d i o n a l a n d u n equal in P. massiliensis, a n d in b o t h species the clear c y t o p l a s m is divided d i s p r o p o r t i o n a t e l y to the blastomere v o l u m e s (t-test: p < 0.01) with the b u l k of the clear

247 Table 1. Mean values of morphometric analysis of the blastomere volumes and their cytoplasmic compositions at the 2-celI stage of Platynereis massiliensis (n = 7) and (in brackets) Platynereis dumerilii (n = 7; from Dorresteijn 1990) Blastomere

Clear cytoplasm

Yolk and lipid

Total volume

AB CD

2.96-+0.55 (7.65)" 8.13 -+1.66 (27.56)b

31.30-+2.89 (19.06) 57.60 -+ 1.84 (45.30)

34.26+2.68 (26.74___0.95%) 65.74 _+2.69 (73.26-+0.95%)

" Data are presented as a percentage of total egg volume b CD blastomere is equipped with 73-77% of the clear plasm of the zygote in Platynereis rnassiliensis and with 78% in Platynereis dumerilii Table 2. Mean values of morphometric analysis of the blastomere volumes and their cytoplasmic compositions at the 4-cell stage of Platynereis massiliensis (n = 6) and (in brackets) Platynereis dumerilii (n = 3 ; from Dorresteijn 1990) Blastomere

Clear cytoplasm

Yolk and lipid

Total volume b

A B C D

1.19__+0.13 (3.94)a 1.92±0.33 (3.71) 2.29_+0.57 (6.52) 6.30-+0.69 (21,04)

11.90--+1.21 (9.01) 18.86-+2.12 (10.05) 18.89-+2.43 (15.44) 38.57-+ 1.43 (29.86)

13.14-+1.15 (13.04+1.64%) 20.77-+2.17 (13.85-+0.76%) 21.18-+2.77 (22.07-+2.08%) 44.90-+ 1.59 (51.04-+1.84%)

Data are presented as a percentage of total egg volume b Note that in P. massiliensis, due to the unequal cleavage of AB, the larger B is nearly equal in size to the C blastomere, but there is no significant difference in size between the A and B blastomere in embryos of P. dumerilii

cytoplasm ending in the larger CD blastomere (Table 1). The plane of first cleavage (Fig. 2) always runs perpendicular to the longest axis of the zygote and in this respect differs from the P. dumerilii zygote in which the position of the first cleavage plane cannot be predicted on the basis of egg geometry. The long egg axis, together with the animal-vegetal axis, thus defines the sagittal plane of the future embryo, even in the uncleared egg. The volume proportions between the AB and CD blastomeres are more variable in P. massiliensis (Table 1).

volumes of the first quartet of micromeres ( l a - l d ) makes up only 4 - 5 % of the total egg volume. Clear cytoplasm, however, is partitioned nearly equally in the A, B and C quadrants. Only in the D quadrant does the bulk of clear cytoplasm remain in the macromere (Table 3). Micromeres 1 a-1 d contain 32-36% of the total amount of clear cytoplasm. All these parameters (1) formation of micromeres and macromeres, respectively; (2) disproportionately high contribution of clear cytoplasm to the micromeres 1 a-1 d; (3) a comparatively high amount of clear cytoplasm remaining in the macromere 1D - are shared by the P. dumerilii embryo.

Second cleavage

As in P. dumerilii, the nuclear events of second cleavage start earlier in the larger CD blastomere, although the difference is only 2 min after an interphase of about 130 rain. As in the 4-cell stage of P. dumerilii, and conforming to the traditional nomenclature for spiralians, the largest blastomere is named D, the others, viewed from the animal pole, are designated A, B, and C in a clockwise fashion (for morphometric data, see Table 2). The embryo of P. massiliensis differs from that of P. dumerilii in that both blastomeres AB and CD divide unequally. As in first cleavage, clear cytoplasm is divided disproportionately among the daughter blastomeres during second cleavage; the largest cell, D, is 43.7 47.8% of the total egg volume, but receives 50 58% of the total clear cytoplasm. Clear cytoplasm is distributed disproportionally in both species.

Third cleavage

Third cleavage is spiral, dextral and extremely unequal, especially in the D quadrant (Fig. 4). The sum of the

Fourth cleavage

The events connected with fourth cleavage in the embryo of P. massiliensis perfectly parallel those in the cleaving eight-cell stage of P. dumerilii. The results of fourth cleavage are shown as histograms in Fig. 5, and a comparison between both species is possible from Table 4. Both quartets of the eight-cell stage divide sinistrally at fourth cleavage. The micromeres 1 a-I d divide into a group of larger blastomeres 1 a1-1 d ~, rich in clear cytoplasm and surrounding the animal pole, and a more vegetally situated quartet of cells, I a 2 1 d 2. The latter are only a quarter of the volume of the mother cell, rich in yolk and are the precursors for the primary trochoblasts. In the macromeres, yolk has compacted towards the vegetal pole before fourth cleavage starts. This causes much of the clear cytoplasm of the macromeres to be segregated into the second micromeres 2 a - 2 d by the extremely unequal cleavage which follows. Since the amount of clear cytoplasm remaining in each of the macromeres 1 A-1 D is different, the size of their micromere

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Table 3. Mean values of morphometric analysis of the blastomere volumes and their cytoplasmic compositxons at the 8-cell stage of Platynereis massiliensis (n = 5) and (in braekets) of Platynereis durnerilii (from Dorresteijn 1990) Blastomere

Clear cytoplasm

Yolk and lipid

Total volume

la lb lc ld IA 1B 1C 1D

0.64--0.07 (2.87)a 0.92_+0.06 (2.95) 1.14-t-0.26 (5.44) 1.43_+0.13 (4.54) 0.55+0.18 (1.59) 1.13_+0.21 (1.19) 1.24_+ 0.40 (2.79) 4.96 _+0.73 (19.54)

0.08+_0.03 (0.53) 0.07-t-0.01 (0.35) 0.09_+0.03 (0.23) 0.03+_0.03 (0.24) 11.69_+1.21 (7.02) 18.80_+2.37 (9.46) 18.58 ± 2.67 (14.69) 38.69 _ 1.67 (26.57)

0.72_+0.08 (3.40) 0.99_+0.07 (3.31) 1.20_+0.24 (5.67) 1.47_+0.15 (4.79) 12.23_+1.21 (8.60) 19.92+2.47 (10.65) 19.82_+ 2.89 (17.48) 43.65 _+1.66 (46.11)

a Data are presented as a percentage of total egg volume

daughter cells 2 a - 2 d , consisting largely of clear cytoplasm, differs extremely. The largest a m o n g the second micromeres is cell 2d, which contains 27% of the total a m o u n t of clear cytoplasm present in the embryo, i.e. twice the volume retained by 2 D (Fig. 5d). The a m o u n t of clear cytoplasm still present in macromeres 1 ~ 1 C is small, and so are the micromeres that are formed by them at fourth cleavage (Table 4). Thus, the 2d cell is nearly four times the size of blastomere 2c, nearly five times that of 2b, and up to eight times the size of blastomere 2a. As in P. dumerilii, the blastomeres of the 16-cell stage are thus already very diversified with respect to their volumes and cytoplasmic composition, as shown in Fig. 5. Blastomere 2d, the "first s o m a t o b l a s t " , will give rise to a large part of the trunk tissues of the larva.

Fifth cleavage As in the embryo of P. dumerilii, the fifth, dextral cleavages of P. massiliensis m a r k the beginning of a pronounced asynchrony a m o n g the blastomeres. Whereas during fourth cleavage the duration of the cell cycles differs by only 2 min (with a cell-cycle of about 172 min) a m o n g the blastomeres, the duration of the cell cycle in the last blastomeres to enter fifth cleavage (about 230 rain, in cells 2 b and 2c) exceeds that of the earliest blastomere (2D) by 84 rain. This interval must be added to" the delays in earlier cleavage cycles, so that the last blastomeres enter fifth cleavage a full 89 min after the earliest. After the completion of fifth cleavage in all cells, the 32-cell stage m a y exist for up to 40 rain. However, in some embryos (Figs. 6, 8), the earliest cell to undergo

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Fig. 5a-d. Histograms of the total volumes of the blastomeres of the 16-cell P. massiliensis embryo ( n = 4 ; a) and of partial volumes of yolk ( n = 4 ; b), clear cytoplasm ( n = 4 ; d) and nuclei ( n = 1; e). The blastomeres are already very diversified with respect to their volumes and to their cytoplasmic composition. The macromeres 2 A 2D are the largest cells and contain the bulk of the yolk. In some embryos, macromere 2B may be somewhat larger

Io I Ib I Ic I Id I la 2 Ib 2 Ic 2 Id 2 2a

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than 2C. Blastomere 2 d ist not only the largest among the micromeres but also contains much more clear cytoplasm than any other cell. Note the positive correlation between the amount of clear cytoplasm in a blastomere and the size of the nucleus. Blastomere 2D of this embryo is entering mitosis and therefore has its nucleus already condensed (asterisk). Vertical lines indicate the variance encountered

25O Table 4. Mean values of morphometric analysis of the blastomere volumes and their cytoplasmic compositions at the 16-cell stage of Platynereis massiliensis (n = 4) and (in brackets) of Platynereis dumerilii (n = 3; from Dorresteijn 1990) Blastomere

Clear cytoplasm

la ~ 1bI lc 1 ld ~ la z lb 2 lc e ld 2

0.496-0.06 0.686-0.04 0.856-0.18 1.11_+0.02 0.156-0.01 0.25-+0.04 0.29_+0.12 0.38_+0.07

2a 2b 2c 2d 2A 2B 2C 2D

0.416-0.04 (1.17) 0.73-+0.04 (0.75) 0.84_+0.19 (1.74) 3.30_+0.64 (16.03) 0.216-0.07 (0.56) 0.46+0.12 (0.42) 0.54+_0.12 (1.10) 1.65 _+0.26 (4.06)

(2.28) (2.09) (3.85) (3.61) (0.99) (0.94) (1.19) (1.27)

Yolk and lipid

Total volume

0.04___0.01 0.02_+0.01 0.03_+0.02 0.01_+0.01 0.05_+0.02 0.06-+0.01 0.05_+0.03 0.03_+0.03

0.52_+0.06 0.69_+0.04 0.88_+0.16 1.12+0.01 0.19-t-0.03 0.31_+0.05 0.34_+0.12 0.40_+0.11

(0.34) (0.26) (0.25) (0.27) (0.25) (0.33) (0.23) (0.29)

0.04+0.01 (0.20) 0.03-+0.02 (0.07) 0.05_+0.01 (0.16) 0.03_+0.05 (0.19) 11.41___1.22 (8.01) 18.53-+2.67 (8.83) 18.64_+3.06 (14.30) 38.70 _+1.95 (23.99)

(2.62) (2.36) (4.10) (3.88) (1.24) (1.27) (1.42) (1.56)

0.44-+0.05 (1.37) 0.76_+0.03 (0.80) 0.89-t-0.19 (1.90) 3.34_+0.67 (16.21) 11.61_+1.30 (8.57) 18.99_-t-2.79 (9.25) 19.18_+3.15 (15.40) 40.35 6-1.96 (28.05)

Data are presented as a percentage of total egg volume l d z2

Fig. 6. Lateral view of a 26-cell stage of P. massiliensis. The animal micromeres l ala-l d ix, l a lzl d lz, l d Z l + l d 22 as well as the micromeres 2d ~, 2d 2, and 3a 3d are products of the fifth cleavage, while micromeres 2 ~ 2 c are still uncleaved. The primary trochoblasts i a z - i c z (all trochoblasts stippled) are undergoing fifth cleavage, while macromere 3D is beginning sinistral sixth division. Note the small size of third micromere 3d. Scale bar, 100 ~tm sixth cleavage (3 D) does so even before the last blastomeres have completed fifth cleavage; the same is always true for P. dumerilii. Blastomere 2 d m a y cleave not much later than macromeres 2 D - 2 A and micromeres 1 d I l a 1, but the moment at which this prominent cell enters fifth cleavage varies to a remarkable degree. In P. dumerilii, cleavage of cell 2d starts at a fixed time (13-15 rain after 2D), which is later than its high content of clear cytoplasm might lead one to suspect (see Discussion). In this and in later stages of the P. massiliensis embryo, the nuclei and the cell cycles of whole embryos could only be analysed after fluorescent staining. Such preparations (Fig. 7) revealed a great deal of diversity in nuclear size and staining intensity (both of them apparently related to the a m o u n t of clear cytoplasm; see Fig. 5) and enabled us to assign the nuclei to pairs of sister nuclei. Figure 7 shows an example of how cell lineage and quadrant kinship can be worked out f r o m both nuclear position and m o r p h o l o g y in whole embryos.

During fifth cleavage, the macromeres again follow different strategies in the A, B and C quadrants in comparison to the D quadrant. Whereas the amounts of clear cytoplasm segregated in 3 a, 3 b and 3c by their respective mother cells are about half the volume of clear plasm available, blastomere 3 d contains only about onefifth of the total quantity of clear plasm initially present in macromere 2 D. The bulk of the yolk-free cytoplasm is retained in cell 3D and is utilized in the formation of micromere 4d. Fifth cleavage is extremely unequal in micromere 2d, cell 2d 2 representing only 10% of the volume of the mother cell. The behaviour of cells 2 A 2 D and 2d during fifth cleavage is very similar to that of identical cells in the P. dumerilii embryo.

E a r l y d e v e l o p m e n t f o l l o w i n g f i f t h cleavage

Sixth cleavage is sinistral and starts with the blastomeres particularly rich in clear cytoplasm, 3 D, 2d 1 and 1 d 11

251

Fig. 7A-D. Nuclei of an embryo of P. massiliensis during fifth cleavage focussed at three different levels (A-C) and a diagram grouping and identifying these nuclei according to cleavage generations and quadrants (D). View from the animal pole. The nuclei are labelled by numbers as follows: 1 = l a11-1 d 11; 2= 1a12-1 d 12; 3 = 1a 2-1 d 2; 4 = 2 a-2 d; 5 = 3 a-3 d; 6 = 3 A-3 D. The living embryo had been incubated with BrdU, fixed, incubated with anti-BrdU and analysed in the epifluorescence microscope. Note the differences in size and fluorescence intensities among the nuclei and the different distances between paired nuclei. Scale bars, 100 gm |

1 a s ~ (Fig. 8), as in P. dumerilii. Sixth cleavage is particularly late in the trochoblasts I a12-1 d 12, 1 a 21 1 d 21 and l a22-1d22 whose cell cycles last more than twice as long as in cell 2d 1 (301-339 and 135 min, respectively). They stop cleaving after the sixth cell division and differentiate as prototroch cells. Contrary to P. dumerilii, a size gradient f r o m the I d 2 to the 1 a2 trochoblasts persists in their descendants, and they, as well as their descendants, never cleave synchronously. Note that the observed minor asynchronies of only a few minutes cannot be read f r o m our cell-lineage diagram (Fig. 3). In the descendants of micromere 2d, the cell cycles get shorter and shorter in the following order: 2d (cell cycle duration 167 rain), 2d t (135 min), 2d 11 (125 rain), 2d 112 (100 min). In this lineage, as in P. dumerilii, one daughter cell retains m o s t of the volume; the respective sister blastomeres 2 d 2, 2 d ~2 and 2 d ~1t are small cells. The macromeres 3 A - 3 D bud off a set of 3 rather small 4th quartet micromeres, 4 a-4 c, and a larger micromere 4 d. Apparently, all the clear cytoplasm which can be spared is shifted into the micromeres. The resulting macromeres 4 A 4 D are stuffed with yolk elements even in the perinuclear region and stop cleaving. During later development they f o r m a large plug in the juvenile w o r m and support its growth until it has acquired 13 parapo-

dial segments and, at an age of 26 30 days, starts feeding. Blastomeres 4d and 2d are particularly large blastomeres (Fig. 9) and contain clear cytoplasm almost exclusively. As in P. dumerilii, cell 4d and a descendant of micromere 2d, cell 2d 112, are situated in the future dorsal midline of the embryo and, by the following cleavage, start to imprint bilateral symmetry on the embryo; seventh cleavage in micromere 4d results in two cells of equal size (4d 1, 4d2), and so does eighth cleavage in cell 2d 112 (2d la21, 2d~122). Thereby the sagittal plane becomes manifest which cuts diagonally through the 1 c 1 d and the 1 a-1 b arms of the "annelid cross", a cross figure at the animal pole built up by the descendants of the animal cells 1 a ~~-1 d 1 t (Fig, 9). The histogenetic role of the mesentoblast (or " s e c o n d s o m a t o b l a s t " ) 4d has not been followed in this species. However, the descendants of cell 2d 112 have the same histogenetic fate as in P. dumerilii: they consist of m a n y rapidly proliferating cells which become displaced towards the ventral midline forming the ventral plate with rows of small neuroblasts and three to four bilaterally symmetrical setal sac primordia. At an age of about 48 h, the trochoblasts develop cilia and the embryo starts rotating around its antero-

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Fig. 8. View of the animal poles of 29-cell-stages of P. massiliensis (below) and P. dumerilii (above left) and of the vegetal pole of a 29-cell-embryo of P. dumeHlii (above right; after Dorresteijn 1990). Note the diffel:ences in relative cell size and, at the same time, identity of ceil pattern and cleavage schedule in the embryos of both species (1 c tl, l d 1~, 2a, 2b, 2c, 2d 1 and 3D in the process of cleavage). The mother cells ( i a t - i d 1) of the secondary trochoblasts (ia12-1d 12) had been unequal in volume in the P. massiliensis embryo, but the cells laX2-Id la are equal to each other and nearly equal with the primary trochoblasts i a2~-I d 2 and l a22-1d 22 (all trochoblasts stippled). Note that the figures are drawn to the same scale. Scale bar, 100 gm

posterior axis. This stage corresponds to the free-swimming trochophore larva of P. dumerilii and may be called trochophoroid stage. Unlike the trochophore of P. dumerilii, the trochophoroid stage of P. massiliensis does not leave the egg jelly nor the brood tube and the young do not escape from the male's care before they have progressed far into postembryonic development. Five to seven days after fertilization, setae have developed in the setal sacs and muscle contractions are detectable, but the trochophoroid is still rotating in its egg jelly. Six to nine days after fertilization, the organs of a young worm have developed, including a head with 2 pairs of eyes, I pair of antennae and peristomial cirri. This stage has left the egg jelly, but its 3-4 pairs of parapodia can hardly be used for coordinated locomotion, since the whole animal appears ballooned by the 4 huge macromeres (Fig. 10 a). After 2 weeks of development, the young seven-segmented worm starts cephalization of its 1st pair of parapodia into head appendages as has already been described for P. dumerilii (Hauenschild and Fischer 1969; Fischer 1985). We were unable to determine the circumstances under which the young abandon the brood tube. Many of them were eaten by the male phase so that only 20 30 young worms out of a brood size of 80 240 embryos may escape his "brood care",

at least under our conditions. At stages of 9 or more parapodial segments, the young worms build a common "canopy" by forming a web of threads glued to the bottom of the glass bowl. Under laboratory conditions young P. dumerilii create similar communal web mats. Once the young P. massiliensis have reached 13 parapodial segments, and have almost used up the yolk in the macromeres, i.e. after about 4 weeks of lecithotrophic development, they start feeding and build their own tubes. In laboratory cultures, worms of this stage, unlike those of P. dumerilii, prefer building new tubes alongside the large tubes of much older individuals.

Reproductive biology P. dumerilii is dioecious and sexual behaviour only starts immediately before spawning after which both sexes are moribund. P. massiliensis is known to be a protandric hermaphrodite (Hauenschild 1951), with male sexual development and behaviour (e.g. brood care) starting early in life. Some days before egg-laying, maturing female phases reinforce their tube with additional web material, giving the tube a brownish-yellow appearance. Smaller worms in male phase from neighboring tubes try to enter

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Fig. 10A. Nine-day-old young worm of P. massiliensis bearing four pairs of parapodia (arrowheads). The giant macromeres contain all the yolk (protein yolk and lipid droplets) and allow development without feeding for several more weeks. The pygidium (below) does not yet bear anal cirri. The head region shows one of the peristomial cirri (p) and a poorly developed stomodaeum (s) but no antennae. B Twelve-day-old young worm of P. dumerilii bearing three

Fig. 9. Animal view of a 46-cell stage of P. massiliensis (below) and a 49-cell-stage of P. dumerilii (above left); above right is the vegetal aspect of the latter. The fast-dividing blastomeres l a l - 1 d 1 have produced 12 cells ( l a l l l - l d 111, lal121-1d 1121 and l a 1122 l d1~22), forming the "annelid cross" surrounded by the girdle of 12 trochoblasts (ia 12 l d lz, I a "-1 l d 21 and I a22-l d22; stippled). The blastomeres 2d 112, protruding deep into the embryo, and 4d lie close to the border between the macromeres 4 C and 4D and will start bilateral-symmetrical cleavages with the next cell divisions, thus defining bilateral symmetry of the embryo and the worm. Scale bar, 100 gm

pairs of parapodia (arrowheads). The yolk is completely used up and the thin-walled lumen of the former macromere compartment is now part of a functional gut, the stomodaeum (s) of which has developed a protrusible pharynx with jaws. The head bears two antennae upend (not in focus) and two peristomial cirri (p). The pygidium carries a pair of anal cirri (not in focus). Scale bar for a and b, 100 gm

254 the tube of the female phase, for some time without success against the defending female phase. As the female phase approaches egg-laying, it becomes less active, and finally one male is found lying side by side with the almost motionless female phase. Sexual interaction resulting in fertilization and egg-laying in the tube seems to happen at or around dusk. After that, the female leaves the tube and may survive for a day or so. An isolated female phase was observed pressing her eggs through intersegmental fissures, collecting them with her palps and fastening them to a thread which she had spun along her body. In the brood tube, the eggs form a monolayer on the sides of a second, thin-walled tube which is now found inside the brood tube and is inhabited by the heavily undulating, pumping male phase. The process of construction of this internal tube has not been observed. The development of the young worm takes about 1 month. During this period the male phase ventilates, defends the brood and removes non-developing embryos. Discussion

In the textbooks, the similarity of developmental patterns among spiralians serves as a paradigm of how embryogenesis can testify to a common ancestry for differing taxa. Nevertheless, there are reports of considerable variation of this spiralian type of development even among closely related forms (Fioroni 1971). The following questions therefore arise: (1) How safely can embryologists use identical nomenclature for the blastomeres in embryos of different taxa without creating a spurious homology (cf Ivanova-Kazas 1981)? (2) How different can the spatial and temporal patterns of development become and yet give similar developmental results ? According to the existing literature (Wistinghausen • 1891; Dorresteijn 1990), the development of P. massiliensis and P. dumerilii seemed extremely different. However, the work of Wistinghausen does not provide enough information to answer such questions, especially because one of us (Dorresteijn 1990) has quantified several parameters of blastomere morphology in the course of early development of P. dumerilii by computer-aided morphometry. Our reinvestigation of the development of P. massiliensis has shown that this new method is a tool to quantify differences between embryos of different species objectively. Quantitative comparison between P. massiliensis and P. dumerilii embryos has revealed differing, species-specific proportions between corresponding blastomere volumes. On the other hand, we can show that the pattern of formation and cytoplasmic specification of quadrants and blastomeres, as well as the pattern of cell cycle durations, follow the same scheme in both species. Thus, we have gathered evidence that the blastomeres in the embryos of these two species become specified and defined by a whole set of congruent topographical, volumetric and cytological parameters, i.e. in a homologous manner. We will first consider the develop-

mental differences we have found between the two species and compare them with those postulated by Wistinghausen; then we will focus on similarities in the two species. The egg of P. massiliensis is more than ten times the volume and, moreover, differs from that of P. dumeriIii in shape and contents. While the egg of P. dumeriIii displays rotational symmetry along the animal-vegetal axis, P. massiliensis eggs are triradially symmetrical ellipsoids, foreshadowing the future sagittal plane of the embryo by the plane defined by the long diameter and the animal-vegetal axis. As Freeman (1982) pointed out, the median plane is frequently obvious sooner in directly developing embryos than in those developing into larvae. Whether such a bilateral symmetry actually is lacking in the egg of P. dumerilii or existing in an undetected way needs, however, to be reinvestigated by an experimental approach. P. massiliensis oocytes contain relatively few cortical granules, whereas they are abundant in P. dumerilii and in other broadcast-spawning nereids. Egg jelly in the latter species promotes floating and dispersal of eggs spawned in the open water, a feature not useful if the eggs are to remain in the parental tube. Most striking, however, is the abundance of yolk in P. massiliensis eggs. Before and during cleavage these yolk components gradually segregate towards the vegetal pole. The amount and asymmetrical distribution of yolk affect the relative sizes of blastomeres. The micromeres towards the animal pole are thus relatively much smaller in P. massiliensis, in which the total volume of the first quartet represents only 4.4% of the total volume (17.2% in P. dumerilii) and in the second quartet only 5.4% (17.8% in P. dumerilii).

The embryos of both species also differ in rate of development; cleavage takes nearly four times as long in P. massiliensis. Observations on free-living nematodes (Skiba and Schierenberg, in press), molluscs (Conklin 1907; Fioroni 1971) and sea urchins (Raft 1987) suggest that, among closely related species in general, yolkier eggs divide more slowly. After standardization of the time scales in Fig. 3, a strong similarity appears between the sequences of cleavages in both species. This shows that the development of all blastomeres of P. massiliensis is nearly four times slower than those of P. dumerilii. This cannot be due solely to the larger size and yolk content of the blastomeres in P. massiliensis, because those at the animal pole are not much larger and are of the same cytoplasmic composition as the corresponding micromeres in P. dumerilii. After standardization, some cell lines of P. massiliensis even seem slightly "accelerated" with respect to homologous cells of P. dumerilii. This concerns the cleavage cycles of the macromeres 3A, 3B, 3 C, the micromeres 2a ~, 2b t, 2c 1, and various cleavages within the 2d strain. If the acceleration of these cell lines relative to others persists (beyond the period of development presented in this paper), it may lead to heterochrony in the differentiation of organ-anlagen. This would create new possibilities for cellular interaction and may influence the schedule by which parts of the body plan

255 are formed (Rensch 1959; Fioroni 1971; Freeman 1982). Since the 2d blastomere is responsible for the formation of the ventral plate and thus forms nearly all neuroectodermal structures of the trunk, the relative acceleration of its progeny is also in agreement with the observations that precursors of adult structures develop more rapidly than precursors of larval tissues in large, yolky eggs (Freeman 1982). This tendency is strongest in those embryos that show direct development and whose larval differentiation is suppressed completely (Raft 1987), but may also be present in intermediate forms of lecithotrophic larvae. The two species of Platynereis deserve a more detailed study of the formation of adult organs. We expected further differences between the embryos of both species comparing our previous observations on development in P. dumerilii (Dorresteijn 1990) with the description of embryogenesis in P. massiliensis by Wistinghausen (1891). However, our analysis of cleavage in both species has revealed that Wistinghausen omitted complete cleavage cycles, notably fourth cleavage in the first micromere quartet and the fifth round of cleavage. Thus, Wistinghausen's description could not be used for our purpose, and our own analysis did not reveal remarkable differences between the two species other than those mentioned above. However, we have established a long list of similarities in embryogenesis between the two species, which we list in the order they occur: - Sorting out of yolk towards the vegetal pole results in a plug of yolk-free, "clear" cytoplasm at the animal pole (" ooplasmic segregation"). First cleavage divides the zygote unequally. During first and many of the subsequent cleavages, clear and yolky cytoplasms are distributed among the daughter cells in proportions differing from the proportions of the blastomere volumes (disproportioning of cytoplasmic components). The second cleavages in the AB- and CD-blastomeres are slightly asynchronous in a fixed sequence, and so are many of the subsequent divisions. - The inequality of first and second cleavage creates size differences among the quadrants ~ D ; this inequality in size is later correlated with differences in cleavage rates and cellular behaviour among the quadrants. During third and fifth cleavages the vegetal sister cells retain half or more of the clear cytoplasm. On the other hand, during fourth and sixth cleavages all the blastomeres which bud towards the animal pole receive more clear cytoplasm than their vegetal sister cells, even if they are much smaller; blastomeres 2d and 4d in the largest quadrant are most conspicuous in this respect. Thus, after sixth cleavage, the cytoplasmic composition of the blastomeres is extremely diverse. As the blastomeres become more and more cytoplasmically diverse, the size of the nuclei become more and more diverse as well. This correlation between the absolute amount of clear cytoplasm and the size of the nucleus is strong and positive. The amount of clear cytoplasm in a cell is in good and positive correlation with its speed of cell cycle; with very few exceptions (e.g. the trochoblasts: see below), -

the blastomeres containing most of clear cytoplasm are those with the shortest cell cycles, and blastomeres 4A4D appearing almost free of clear cytoplasm even stop cleaving. Bilaterally-symmetrical cell division, which imposes bilateral symmetry on the whole future posttrochal region of the embryo, starts in the seventh cleavage cycle in blastomere 4d, the mesentoblast, and in the eighth cycle in blastomere 2d 112, a descendant of the somatoblast 2 d. Thus, apart from more trivial similarities such as the formation of micromeres and macromeres by means of alternating spindle positions, i.e. common features of all spiralians, the courses of early development in P. massiliensis and P. dumerilii are in excellent agreement. Differences, though appearing conspicuous at the first glance, are restricted to differing proportions in temporal and spatial pattern and may be due to drastically increased yolk content. In addition to differences in early development, a whole set of remarkable differences between P. dumerilii and P. massiliensis has been observed with respect to reproductive biology by the present and earlier authors (Hauenschild 195/; Pfannenstiel etal. 1987; Lficht 1987). In P. dumerilii, both sexes are broadcastspawners, shedding their gametes after an epitokous metamorphosis during a vigorous nuptial dance in the open water, whereas P. massiliensis reproduces benthonically and as a protandric hermaphrodite. The question therefore arises as to which styles of reproduction and early development can be considered more primitive and which evolutionary step may have been the driving force for the evolution of one or other Platynereis species. In P. dumerilii, eggs develop while floating in the open water into free-swimming, positively-phototactic trochophorae, suited for dispersal of the brood; early juvenile worms (" nectochaeta larvae") move freely by swimming and crawling. Early stages of P. massiliensis are deficient in these respects; the egg is covered by some egg jelly but cannot float, the trochophoroid has a ciliary belt but cannot swim, and the three-segmented young have parapodia but cannot crawl coordinately. Therefore, floating eggs and free-moving larvae and juveniles seem primary characters, whereas benthic development and extremely yolky eggs seem secondary traits. Slow, benthic development without protective envelopes for the brood, however, necessitates development to occur inside the parental dwelling tube and/or under parental care, as found in P. massiliensis (and other nereids; cf. Weinberg et al. 1990). Benthic reproduction inside the dwelling tube therefore must have preceded the evolutionary process leading to heavy, yolky eggs with moderate egg jelly layers, and to loss of locomotive function of both larval cilia and the earliest parapodia. The latter structures in P. massiliensis are thus explained as conserved developmental traits of an ancestor passing through embryonic and larval stages resembling those of P. dumerilii. Our present results have shown that almost every step in every cell line and every temporal and spatial pattern of diversification among the growing number of blasto-

256 meres is r e t a i n e d in b o t h species o f Platynereis, even t h o u g h the e m b r y o s initially seemed to be very different. C o m p a r a t i v e e m b r y o l o g y , p a r t i c u l a r l y if s u p p o r t e d b y m o r p h o m e t r y , thus p r o v i d e s evidence t h a t n o t o n l y principal events a n d p a t t e r n s b u t even details o f early spiralian d e v e l o p m e n t m u s t n o t be c h a n g e d if the results o f d e v e l o p m e n t are to be similar.

Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft (Bonn, FRG; grants Do 339/1-I and Do 339/1-2). We thank the Feldbausch-Stiftung of the University of Mainz and the staff of the Laboratoire Arago at Banyuls/France for supporting our effort to build up a laboratory stock of P. massiliensis. We are grateful for the advice of Prof. Pfannenstiel and Dr. J. Liicht (both Berlin) in the matter of maintenance. The critical reading of the manuscript by Dr. P. Schroeder (Pullman, USA) and Daniele J6rg (Mainz) is gratefully acknowledged. We thank Mrs. K. Rehbinder for helping with the graphical design, Mrs. M. Ullmann for help in preparing the photographic plates and Mrs. M. Eberts for typing the manuscript.

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