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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 µm) 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 µm 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 10µm
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.
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Cluster
of symplasmic oocytes
surrounded by somatic sheath cells (from Fischer 1975)
Unfertilized oocyte (see text)
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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.
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The tapered acrosomal process starts contact with
one of
numerous microvillar tips projecting through the vitelline
envelope of a Platynereis oocyte
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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 18°C 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.
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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)
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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 µm; length of the juveniles (L – O):
300 µm.
From Fischer & Dorresteijn (2004) BioEssays 26: 314-325.
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 trunk of the trochophore larva
forming a Y-shaped configuration. D =
dorsal ; V = ventral
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