Development of Platynereis dumerilii

 

 

by Adriaan Dorresteijn & Albrecht Fischer

 


1. Gametogenesis

As in many other organisms, several developmental steps in the early embryo are prepared during gametogenesis, i.e. well before fertilization. The location of neither the germ line nor the primordial germ cells is known in nereidids. Since the gametocytes grow and differentiate while floating freely within the coelomic cavity, gametogenesis can be studied under favorable conditions both in vitro and in vivo.

1.1 Oogenesis

Ultrastructural studies of oogenesis by Fischer (1975) revealed that clusters of small (<27 m) previtellogenic oocytes initially stick together due to incomplete cytokinesis of sister cells. Each of these clusters is surrounded by thin somatic sheath cells. During the phase of vitellogenesis the oocytes increase in size and break away from the narrow confinement of the cluster. The yolk precursor called vitellogenin is produced by other coelomocytes, the eleocytes. The kinetics of vitellogenin uptake (Fischer & Rabien 1986, Fischer et al. 1991) speak for a receptor-mediated process. At the end of oogenesis the oocyte measures 160 m in diameter. The central nucleus (a germinal vesicle still in Prophase I of meiosis) is surrounded by randomly distributed yolk granules, lipid droplets, strands of the endoplasmic reticulum, ribosomes, and small cortical granules. Large dictyosomes take a position immediately underneath the oocyte surface lined by numerous microvilli. During the last 36h before spawning the oocyte completely reorganizes its cytoplasmic constituents. A 10m thick layer of cortical granules now underlies the oocyte surface. The number of microvilli is dramatically reduced. Large lipid droplets become surrounded by tightly packed yolk granules and seem pressed against the cortical layer. A distinct concentric layer of perinuclear cytoplasm (clear cytoplasm) is formed around the germinal vesicle (Rosenfeld, unpubl.).

1.2. Spermatogenesis

Spermatogenesis starts with primary clusters of spermatogonia (Pfannenstiel et al. 1987). The actively dividing cells remain connected by cell bridges. Eventually, the clusters disintegrate, producing secondary clusters of spermatogonia. Following numerous rounds of nuclear division such clusters fall apart into spermatocyte clusters. Spermatocyte nuclei are identified as such by the presence of synaptonemal complexes. The cells of these clusters show globular cellular inclusions, the proacrosomal vesicles. In the last couple of weeks before spawning, the spermatocytes develop into spermatid tetrads which start spermiogenesis. During the last four days before spawning, the spermatid tetrads separate from the clusters and finally fall apart into single sperm. The release of the sperm during a nuptial rendez-vous is accomplished by a sperm sprinkler (the pygidial rosette) in the modified pygidium. The sperm is of the primitive type with a small middle piece, a round head and a tapered acrosome.

Oogenesis
Cluster of symplasmic oocytes surrounded by somatic sheath cells (from Fischer 1975)


unfertilized egg
Unfertilized oocyte (see text)




2. Fertilization, cortical reaction and ooplasmic segregation


Under natural conditions fertile males and females of Platynereis swim to the sea surface on dark nights around new moon. The swarming individuals shed pheromones (5-methyl-3-heptanone; 3,5-octadien-2-one; uric acid) essential for gamete release during the nuptial dance (Boilly-Marer 1969; Boilly-Marer and Lasalle 1987; Zeeck et al., 1988, 1991,1998). The gametes meet in and are diluted by the ambient sea water. Dilution reduces the chance of polyspermy. Under such conditions only a few sperm reach the egg surface.
Once the fertilising sperm attaches to the tip of one of the numerous microvilli (penetrating the vitelline envelope) the egg cortex reacts by the release of the cortical granules (Kluge et al. 1995). The discharged contents swell forming the egg jelly. This egg jelly also drives supernumerary sperm from the egg surface, thus helping to prevent polyspermy. This cortical reaction lasts approximately 25 min and is followed by several waves of cortical contraction running from the animal to the vegetal pole. These contractions coincide with the movement of yolk granules and lipid droplets towards the future vegetal pole, which has been called ooplasmic segregation. Approximately 45 min after fertilization the sperm nucleus enters the egg cytoplasm through the narrow cytoplasmic bridge produced by the fusion between the tip of the acrosomal process and a microvillus. (
MPEG-film; 7,7 MB).

Under laboratory conditions fertile males and females can be put together in small glas bowls with 50-100 ml of natural sea water (see the section on Breeding). To avoid polyspermy the supernatant of the egg batch containing the sperm should be removed by a pasteur pipette within a minute after gamete release. Afterwards the eggs should be redispersed in fresh natural seawater. Polyspermic egg batches will divide irregularly and never form normal trochophores.

Sperm on oocyte surface
The tapered acrosomal process starts contact with one of
numerous microvillar tips projecting through the vitelline
envelope of a Platynereis oocyte


3. Formation of the polar bodies and karyogamy


Upon fertilization the egg is released from meiotic arrest. At 18C, , the germinal vesicle breaks down seventeen minutes after fertilization and spindle formation sets in. Between 30 and 45 min after fertilization the spindle attaches to the cortex of the future animal pole. The start of polar body formation is heralded by the flattening of the animal pole, producing a wide gap between the egg surface and the vitelline envelope. The first polar body is pinched off around 60 min, the second polar body around 80 min after fertilization. Subsequently, the maternal pronucleus forms and rounds up within the yolk-free animal pole plasm 20 m beneath the egg surface. The paternal pronucleus migrates towards the maternal pronucleus from a vegetal position. The close apposition of the pronuclei is instantaneously followed by fusion (karyogamy). The zygote nucleus rounds up and becomes indented by the asters of the first mitotic spindle. Nuclear envelope breakdown occurs approximately 100 min after fertilization.

pronuclear fusion
Section parallel to the egg axis showing the animal pole
plasm with the maternal (upper) and paternal (lower)
pronucleus shortly before fusion.


4. The process of development up to the trochophore larva


The early development of the Platynereis embryo can be divided into three distinct phases. The first phase is characterized by a strictly unequal spiral cleavage pattern. The cellular nomenclature for the spiral cleavage used here conforms to that proposed by Conklin (1905) in his description of the development of the snail Crepidula. The second phase is characterized by the equal and bilaterally symmetrical cleavage of the trunk-forming cells, the somatoblast (2d) and the mesentoblast (4d). So far, the offspring of all cells have remained in their original positions. But during the third phase of early development, epibolic gastrulation movements set in and the 2d- and 4d offspring start moving in a lateral or posterior direction.

4.1 Phase one: unequal spiral cleavage
At 18C  the formation of the first cleavage furrow sets in 110 min after fertilization (p.f.) and cuts the egg cytoplasm from the animal pole towards the vegetal pole. Due to the unequal size of the asters, the mitotic spindle takes up an asymmetrical position within the animal pole plasm. As a result, the first cleavage cuts the egg into blastomeres of unequal size. The larger CD-cell (73% of the total egg volume) and the smaller AB-cell (27%) also inherit disproportional amounts of the animal yolk-free cytoplasm (80 % and 20%, respectively). The second cleavage starts 30 min after the first, but nuclear breakdown in the CD-blastomere occurs 1 min ahead of the same nuclear event in the AB-blastomere. The latter cell divides equally, whereas the CD-cell divides into a small C- (22% of the total egg vol.) and large D-blastomere (51%). Again, the distribution of the yolk-free cytoplasm is disproportional so that 60% of the yolk-free cytoplasm of the egg ends up in the D-blastomere. The blastomeres A, B, C and D are founder cells of the four embryonic quadrants. At third cleavage each of the quadrants forms a micromere towards the animal pole of the egg. Due to the oblique position of the cleavage spindle with respect to the animal-vegetal axis the micromeres 1a, 1b, 1c and 1d form dextrally, i.e. in a clockwise direction if viewed from the animal pole perspective. At the following fourth cleavage a second quartet of micromeres is formed in a laeotropic spiral cleavage, giving rise to the blastomeres 2a, 2b, 2c and 2d. In contrast to the previous cleavage, the cell 2d - the second micromere within the D-quadrant - is exceptionally large (approximately 15% of the entire egg volume), contains only a few yolk granules, and marks the future dorsal side. Almost simultaneously, the first quartet of micromeres divides by a laeotropic spiral cleavage into four animal sister cells (1a1-1d1) and four "vegetal" sister cells (1a2-1d2). The latter blastomeres are the primary trochoblasts and will participate in the formation of the prototroch of the trochophore larva.
The third quartet of micromeres (3a-3d) is given off by a dexiotropic spiral cleavage approximately 240 min p.f.. The cell divisions of the blastomeres in the progeny of the first and second quartet lag behind, so that the completion of the fifth cleavage takes about 40 min and overlaps with the sixth cleavage.
At sixth cleavage a particularly large micromere (4d) forms within the D-quadrant by a laeotropic spiral cleavage. Like 2d, this is a large cell, almost devoid of yolk granules, and takes a position on the dorsal median of the embryo. After the formation of the 4d-cell the other blastomeres require up to 80 additional minutes to complete the sixth cleavage cycle. The fate map shows that the ectoderm of the head is basically formed by the progeny of 1a1-1d1; the prototroch is formed by 1a2-1d2 (complemented by cells which arise from 1a12-1d12); the ectoderm of the trunk is formed by 2a-2d and 3a-3c; the primary mesoderm stems from the 4d-cell; the endoderm is formed by 4a-4c, by two small cells in the progeny of 4d, and by the macromeres 4A-4D.


4.2 Phase two: bilaterally symmetrical cleavage of 2d and 4d

The blastomeres 2d and 4d are exceptionally large, almost yolk-free blastomeres. They are given off  by a laeotropic cleavage in the 4th and 6th division cycle, respectively. Their position marks the future dorsal midline.
The fate of the 2d-cell lies in the formation of the major share of the ectoderm of the trunk, of the setal sacs, and of the ventral nerve chord. For this reason the 2d-cell is called the somatoblast. Its fate map has been confirmed by dye injection experiments by Ackermann (2003). To accomplish the bilaterally symmetrical pattern of the setal sacs, and to reach the ventral midline, the 2d-cell proliferates rapidly in a bilaterally symmetrical pattern. Initially, three small cells are given off to the posterior right side (2d2), to the posterior left side (2d12), and, along the midline, to the anterior (2d111). At the subsequent eighth cleavage, the 2d112-cell divides in perfect bilateral symmetry forming the progenitors of the left (2d1122) and right (2d1121) side of the trunk ectoderm. All the subsequent cleavages occur in almost perfect mirror symmetry on either side of the dorsal median. The dorsal accumulation of the 2d-offspring disappears gradually during epiboly - a mode of gastrulation by which the circumference of the yolky macromeres is slowly covered by these ectodermal cells.
As described in the previous part, the 4d-cell is the founder of the trunk mesoderm (and was believed to contribute to the endoderm as well) and was therefore called the mesentoblast. The cleavages of this particular cell are in perfect bilateral symmetry from the start. From the seventh cleavage onwards the 4d1-cell forms the left, the 4d2-cell the right mesodermal germ band. The germ band progenitor cells are overgrown by 2d-offspring at the posterior blastopore rim during epibolic gastrulation. The mesodermal germ bands initially grow by lateral cell division and surround the posterior tip of the 4D-macromere on either side. The germ bands meet at the ventral plate and send small cells towards the stomodaeum. In the trochophore larva the mesodermal germ band therefore appears in a Y-shaped configuration.
.
4.3 Phase three: epibolic gastrulation

The driving force for epibolic gastrulation seems to be the massive cell proliferation by the somatoblast and the mesentoblast. Basically, the progeny of the 2d-cell move from a dorsal position into a latero-ventrad direction. The posterior dorsal rim of the epibolic movement also extends towards the vegetal tip of the 4D-cell and overgrows the anlagen of the mesodermal germ bands. At about 16h p.f. the lateral rims of the blastopore meet at the ventral midline. The offspring of the other second and third quartet micromeres are driven together in a ventral triangular region immediately posterior to the prototroch. In this region the stomodaeum invaginates.

Further details of early development can be found in Dorresteijn (1990)


early development

A – D Ooplasmic segregation and diversification of
quadrants in the embryo. E – G Spiralian mode of cleavage and its transition (H) from a radial to a bilateral symmetric cleavage pattern in some cell lines. I – L Larval and post-
larval development from the trochophore (I) to the three-
segmented young worm (“nectochaete”) of three (K) and
four days of age (L). M – O Anatomy of the three-
segmented five-days-old juvenile,schematized,  dorsal
view, with the central nervous system (yellow) in M, plus musculature (red) in N and gut anlage (green) in O.
Explanations: (A) asterisk: animal pole; large circles: lipid; small circles: yolk bodies (B) note the asymmetric
distribution of clear cytoplasm between the two blasto-
meres (C – H) 2-, 4-, 8-, 16-, 49- and 66-cell stage , letters and numerals conforming to the standard nomenclature
of spiralian embryos, in (G) restricted to the A-quadrant
delineated by bold lines, green: cells of the future ciliated
belt (I – K) ventral views of a one-day trochophore, a 2-
day metatrochophore and a 3-days-old juvenile: A
antennae, AC anal cirri, AT apical tuft, GC larval gland
cells, LA larval eyes, NR neurogenic region, P prototroch, Pa palps, S stomodeum, SS setal sacs (L) Five-days-old
juvenile. Note the two pair of pigmented adult eyes, the
pharyngeal cleft, lipid drops in the midgut anlage,
elongated peristomial and anal cirri and the pigment cells. (O) Note the subdivision of the gut anlage into pharynx
(the former stomodeum), the solid midgut anlage and the
hindgut.- All embryos (B – H) to scale; diameter of the
early stages: 160 m; length of the juveniles (L – O):
300 m.
From Fischer & Dorresteijn (2004) BioEssays 26:
314-325.

 

bilateral pattern
Dorsal aspects shortly after the division of the 4d- and 2d112-cell from a vegetal view at two different planes of focus showing the bilateral symmetry in their offspring on either side of the dorsal median.


mesoderm band in the trochophore larva
Mesoderm band in the trunk of the trochophore larva forming a Y-shaped configuration.
D = dorsal ; V = ventral

 


5. The trochophore larva (24h)


Since the Platynereis embryo is well-furnished with protein and lipid yolk, it develops into a swimming, yet lecithotrophic larva. The main lipid stores are located within the four macromeres. After concentrating the many lipid droplets of the oocyte in a two-step process during structural reorganization in the late oocyte and in the early macromeres of the embryo, each macromere now carries a single lipid droplet only (the largest droplet is located within the 4D-cell). The development of a continuous gut lumen is postponed for about 7-9 days.
The trochophore larva is spherical and can be subdivided into three regions. The equatorial region is characterized by a girdle of 24 large ciliated cells that form the prototroch.
The episphere (anterior to the prototroch) carries the apical tuft in the position of the apical organ (the larval brain). On either side of the apical organ lie the anlagen of the cerebral ganglion and the optic fields. The latter are characterized by a small larval eye (ocellus). The ventral region of the episphere ectoderm contains the anlagen of the palps and antennae. A group of five larval gland cells lines the ventral border of the episphere. The dorsal ectoderm of the episphere is a thin squamous epithelium.
The hyposphere is characterized by three pairs of ectodermal invaginations. These are the setal sacs from which the parapodia of the deutometameres (= larval segments) will develop. Posterior to the last pair of setal sacs lies the anlage of the pygidium. On the the lateral border of the pygidium lie the short cilia  of the paratroch. The ventral epithelium of the hyposphere is thick and represents a neurogenic region, i.e. the anlage of the ventral nerve cord. Close to the prototroch lies the invaginated stomodaeum. The dorsal ectoderm is a thin squamous epithelium.
The trochophore larva hatches from the egg jelly approximately 20-24h p.f., but never hatches from the vitelline envelope which has been transformed into the larval cuticle. Free-swimming trochophores show a positive phototactic reaction which can easily be used to collect them from bowls of sea water in the laboratory.

trochophore larva
Posttrochal region of the Platynereis trochophore showing
the lateral setal sacs (white arrow heads). The white dashed line shows the ventral midline. The stomodaeum (S) lies immediately posterior to the prototroch.
Posterior pole at the bottom of this picture.


6. The metatrochophore larva (48h)


As does the previous stage, the metatrochophore moves predominantly by means of the cilia of the prototroch and still follows a swimming life-style. Whereas the size of the episphere has hardly changed, the hyposphere has elongated, resulting in the pear-shape of this larval stage. After two days groups of bristles, each formed by a chaetoblast inside the setal sacs, project to the exterior on either side of the deutometameres. The posterior border of each larval segment is lined by 12 ciliated cells (six on the dorsal and six on the ventral side). The optic fields within the episphere shows the anlagen of the two pairs of adult eyes, which lie 2-3 cell diameters in a medio-dorsal direction from the larval ocelli. Larval and adult eyes are separated by a row of ciliated cells. The stages of transition from the metatrochophore to the 4-days-old juvenile worm are illustrated in Fischer (1971) and Hauenschild & Fischer (1969)

 


7. The three-segmented young worm (96h)


After about four days the young worms have three pairs of distinct parapodia, the muscle groups of which allow the animal to crawl. However, the crawling is frequently interrupted by phases of swimming. The swimming activity is now based on the cilia on the segment borders and between the larval and adult eyes. The cilia of the prototroch are gradually disappearing. In this region, two short lateral peristomial cirri are being formed. There is substantial evidence that these first peristomial cirri are appendages of a segment which has been cephalized during evolution (Fischer 1999; Ackermann et al. 2005). Ventro-lateral to the apical tuft, a pair of antennae develops. The five larval gland cells have moved together in the median ventral region between the anlagen of the palps. The posttrochal stomodaeum has developed into a muscular, protrusable pharynx carrying the anlagen of a pair of chitinous jaws. The midgut is still loaded with protein yolk granules and large lipid droplets and has no lumen yet. At the posterior end a small ectodermal hindgut anlage is being formed. The pygidium shows short anal cirri.
The first of the three pairs of parapodia at this stage differs from the second and third pair because it lacks a conical notopodial lip and a parapodial gland. The parapodial glands produce a silky product used by the animals to build a canopy (young stages) or a tube in which they live (older stages).

three-segmented young worm


8. The process of cephalization (starting at 21-28 days)


When, after 3 – 4 weeks Platynereis has reached the stage of a 6-setiger young worm, the first pair of parapodia undergoes a dramatic metamorphosis. Dorsally of the setae a pair of long cirri is formed, later followed by a ventral pair, while the setae are shed (Fischer 1999). These two pairs of appendages shift towards the head and join two more pairs of "peristomial cirri" which have developed at the head without a parapodial precursor. Tracer injection into early blastomeres has shown that all of the peristomial cirri together with all of the trunk ectoderm and mesoderm are derived from the 2d and 4d cell pedigree domains, further attesting the conclusion that all of the peristomial cirri are of segmental, parapodial origin (Ackermann et al. 2005). The process by which a trunk segment is tranformed into a head segment is called cephalisation

six-segmented young worm
The first deutometamere of this six-segmented young worm is in the process of cephalisation. A dorsal appendage (8) is growing and the setae (10) are gradually shed. From Hauenschild & Fischer (1969)


9. Segment proliferation and regeneration in the atokous worm


As do other polychaetes, Platynereis grows by means of segment proliferation. The cellular material for the segment anlagen is produced by a proliferation zone immediately anterior to the pygidium. Should the worms lose their rear end - due to predation which is not a rare event - the wounded stump will form a posterior blastema from which a new pygidium and a proliferation zone develop. The more segments are lost, the faster these segments are regenerated (Hofmann 1966). The regeneration is driven by (a) factor(s) from the prostomium. Removal of the prostomium prevents caudal regeneration and implantation of a prostomium into a truncated worm rescues the ability to regenerate. Segment proliferation and the ability to regenerate are lost as soon as the animal commits itself to its reproductive phase of life by speeding up oogenesis and spermatogenesis.
Regeneration of anterior segments is not possible in Platynereis.

 

 


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last update: 15.03.2004