Sea urchin. Fertilization and cleavage. Normal speed. Time-lapse.

The sea urchin egg is characterized by the homogeneous distribution of deutoplasm, and thus it is particularly suited for observation of total and equal cleavage. To the right, an immature, still diploid ovum with large nuclear vesicle and nucleolus.

To the left, a mature haploid egg with a poorly defined nucleus. The spermatozoa have three distinct morphological regions. The conically pointed head with the acrosome and nucleus, the middle piece with the centrosome and numerous mitochondria, and the tail piece acting as a flagellum.

The sperm swim actively towards the egg cells. The first spermatozoan to reach the surface of the egg penetrates it immediately. At this moment, the ovum is blocked for further sperms. Starting from the point of sperm attachment,

the vitelline membrane rises from the egg to form the fertilization membrane. Cortical granules flow into the fluid-filled perivitelline space, which surrounds the egg after about a minute.

Here, a sperm is just entering the egg. When the fertilization membrane rises from the surface, the egg cortex contracts at the point of entry, showing a temporary indentation. Under the interference contrast microscope, karyogamy is rendered visible. First, the sperm centrosome in the form of an astrosphere

approaches the female pronucleus, here from the left. Soon, the male pronucleus becomes visible. Both pronuclei migrate towards each other, the male pronucleus covering the greater distance. About 40 minutes following sperm entry, the two pronuclei fuse to form the cincarion.

The next stage of development, the prophase, starts with the division of the centrosomes. As the two daughter centrioles separate, a cytoplasmic configuration described as the streak appears, which later gives way to the spindle fibers, the so-called amphiaster, during metaphase.

The first cleavage furrow is formed perpendicular to the spindle axis. Time-lapse cinematography demonstrates the cleavage of four cells up to blastulation. The morphogenesis of the echinoderm egg is characterized by its total and mainly equal cleavage.

The divisions take place at more or less regular intervals, so that after about five hours, eight divisions have been accomplished, resulting in a hollow sphere consisting of 256 blastomeres.

Cilia are developed round the periphery, and the blastula rotates within the fertilization membrane. About seven hours after insemination, the fertilization membrane splits, releasing the blastula to a planktonic mode of life.

The first cleavage is under stronger magnification. The first two cleavage planes are meridional, the third equatorial. Four animal and four vegetal blastomeres are differentiated. The fourth, partly unequal cleavage

gives rise to four micromeres at the vegetal pole. The individual tiers of cells now surround a spherical cavity called the blastocoel. Starting from the fertilized egg, morphogenesis is now demonstrated schematically

up to the 64 blastomere stage. The sea urchin egg exhibits polar symmetry, the animal and the vegetal areas being orientated in graded polarity. The coloring, yellow for animal tendency, green and red for vegetal tendency. This will serve to indicate

the subsequent fate of various prospective areas. The regular cleavage pattern of the sea urchin zygote is due to a characteristic change in orientation of the mitotic spindle plane to the polar axis of the egg. At the first cleavage, the spindle is horizontally aligned on the equatorial plane of the zygote.

So the first cleavage is meridional and leads to the segmentation of equally sized blastomeres. Before the onset of the second cleavage, the two spindles are again aligned on the equatorial plane, but now they are turned at 90 degrees to the plane of projection.

The second cleavage, like the first, is meridional and equal. It results in a tetrad of four equivalent blastomeres. Before the onset of the third cleavage, the spindle axes are vertically orientated, that is perpendicular to the equatorial plane. The third cleavage is affected equatorially

and produces the eight blastomere stage, in which, for the first time, animal and vegetal cell material is separated by cell membranes. The heterogeneous differentiation of the two polar tiers of cells is made evident by their varying cleavage planes. Before the fourth cleavage, the spindles of the animal mesomeric tier are horizontally orientated.

The spindles in the vegetal macromeric tetrad, on the other hand, are eccentric and pointing obliquely to the vegetal pole. The fourth cleavage results in the 16-cell stage. By equal and meridional cleavages,

the animal pole has now differentiated a tier of eight equally sized mesomeres. In the vegetal hemisphere, the specific spindle alignment has conditioned an unequal, approximately horizontal cleavage plane. Thus, the vegetal hemisphere consists of an equatorial tier of four large macromers,

from which four considerably smaller micromiers have segmented towards the vegetal pole. The following cleavages take place alternately on the equatorial and the meridional planes. Thus, at the fifth cleavage, the eight animal mesomeres are divided by equatorial cleavage

into two tiers of almost equal size. The four macromers, conversely, divide meridinally, resulting in a vegetal tier of eight equally sized cells. On account of a somewhat retarded micromier cleavage, the early blastula passes through a 28-cell stage

before reaching the actual 32-cell stage with eight micromiers. At this stage, the early blastula is differentiated into four regions, the two animal cell tiers 1 and 2, the vegetal macromier tier, and a total of eight micromiers at the vegetal pole. The sixth cleavage is again in two phases,

resulting in a 56- and a 64-cell stage. Now the position of the two animal tiers of cells becomes irregular. They ultimately form a rounded cap. Also in the vegetal hemisphere, by horizontal cleavage of the eight macromiers,

both vegetal tiers of cells 1 and 2 have been differentiated. As cleavage proceeds, the original size difference between macro and micromiers is gradually equalized by decelerated division of the micromiers.

These shots show the developmental stages in real life. During the fourth cleavage, that is, from the eight to the 16-cell stage, the four micromiers are in course of differentiation,

here to the right. The subsequent fifth cleavage follows that of the micromiers after an interval. The rhythmical movements of the blastomeres are due to the almost synchronous cleavages. As it proceeds, the blastocele becomes more and more prominent.

The micromiers are no longer visible. Following the eighth cleavage, that is, upon reaching the 256-cell stage, the cilia are formed and the blastula begins to rotate within the fertilization membrane.

The release of the blastula is filmed in three phases at normal speed. The blastula produces a hatching enzyme, which partially dissolves the fertilization membrane so that the embryo can escape.

The blastula is flattened at the vegetal pole and swims with the apical end pointing forwards.

Sea urchin, gastrulation and larval stages, normal speed,

time lapse. After the cleavage divisions of the sea urchin egg follows the transformation of the blastula to the gastrula. To observe this process, the specimens had to be immobilized and the cilia movement blocked. Gastrulation begins with the migration of micromier material,

that is, cells from the central area of the vegetal pole. Within the blastocoel, these differentiate to primary mesenchyme cells while producing protoplasmic processes. They spread out apically by creeping along the walls of the blastocoel. Not long afterwards,

the invagination of the archenteron begins. First, a schematic representation of the development from blastula to gastrula. The totally ciliated blastula has escaped from the fertilization membrane and has become flattened at the vegetal pole.

At the animal pole, a tuft of cilia projects from an epithelial thickening. The blastula is a relatively stable hollow sphere with an interior cavity, the blastocoel, filled with gelatinous substances. The animal germ layers 1 and 2,

which originated in the cellular tiers of the 32 and 64 blastomere stages, are colored yellow. The vegetal tiers 1 and 2 are colored green. The red area indicates the micromiers, which are displaced into the blastocoel before the onset of gastrulation.

Here, in the region of the flattened vegetal pole, they are differentiated into the primary mesenchyme cells with their long protoplasmic processes. Gastrulation begins with the invagination of the material denoted as vegetal 2.

The infolding gives rise to the primitive intestine cavity, the archenteron, the external opening of which is the blastopore. Before archenteron formation has been completed, secondary mesenchyme cells are already migrating to the roof of the archenteron in the blastocoel. At the base of the intestine,

the primary mesenchyme cells are already laying down the rudimentary larval skeleton in the form of three-pointed spicules originating from calcareous crystals. Here, once again under higher magnification, the migration of the primary mesenchyme cells

from the thickened polar plate. The cells issue from the vegetal pole as if they were being subjected to pressure, and they are then displaced into the blastocoel in disorderly fashion.

The archenteron folds itself into the blastocoel. When it is about one-third invaginated, the secondary mesenchyme cells migrate from the roof of the primitive intestine. These mesenchyme cells also exhibit amoeboid movements and are provided with filamentous processes with which they attach themselves to the walls of the blastocoel,

gastrulation viewed this time from the blastopore side. Between the walls and the centrally located archenteron roof are the primary mesenchyme cells that have already migrated.

As the archenteron invaginates, the secondary mesenchyme cells emerge from the now deeply depressed archenteron roof. During the course of gastrulation, the appearance of the larva is changed by a flattening in the area of the presumptive stomodeum.

This can be seen more clearly as the larva is tilted. It is gradually assuming bilateral symmetry. From particular aggregations of primary mesenchyme cells the three-pointed skeletal spicules are secreted. They form the rudimentary calcareous skeleton of the sea urchin larva.

During the late gastrula stage, two mesodermal outpocketings appear on either side of the archenteron roof. They are known as the left and right coelomic sacs.

The first skeletal spicules are produced at the centre of a triaxial syncytium. First, a grain of inorganic matter is laid down. The shape of the syncytium determines the accretion pattern of further layers until the characteristic tri-radiated spicules are recognisable. There is one skeletal rudiment on either side of the archenteron in the blaster seal.

The longitudinal growth of the spicules can be followed very clearly at this late stage.

The differentiation of the coelome, the secondary body cavity from the two mesodome folds at the roof of the archenteron, completes gastrulation. The larva has meanwhile assumed its characteristic prismoidal appearance. The transformation of the late gastrula to a young pluteus larva

is demonstrated schematically. The late gastrula grows faster dorsally than ventrally, thereby taking on a prismoidal appearance. Two mesodermal outpocketings bud off from the archenteron roof,

the left sac developing more noticeably than the right. The archenteron becomes differentiated into three regions by constriction, the esophagus, the stomach, and the intestine opening at the blastopore. Viewed laterally, the more active dorsal growth is apparent.

The archenteron roof arches down towards the future ventral aspect, the animal part of which, coloured yellow, will form the so-called oral field. An oral depression appears, which soon merges into the archenteron roof.

In the young pluteus larva, the oral field is girdled with ciliated bands. At the point of contact between the oral depression and the esophagus, the definitive mouth is now formed. The opposed blastopore now becomes the anus. The three regions of the larval intestine can now be recognised more clearly.

The divisions of the archenteron can now be seen very clearly at this prism stage. To the left is the esophagus, followed by the stomach, and on the right the intestine, emerging at the blastopore.

The skeletal rods now extend along the whole length of the larva. The oral depression now unites with the archenteron roof. Then the definitive mouth opens up, while the blastopore becomes the anus. This process is characteristic of the morphogenesis of the deuterostomia.

These young pluteus larvae already have the rudiments of the four processes which delimit the oral field. In older pluteus larvae, these rudiments extend as arms.

From the side, the interior organisation of the intestine is clearly visible. Mouth, esophagus, stomach, intestine, and anus. The two dorsal arms are almost parallel, being connected by an ectodermal lamella.

The two opposed ventral arms are free and divergent. In this roughly four-day-old pluteus larva, the inner processes leading to metamorphosis have already been initiated.

Not until five or six weeks have elapsed is the development to the young sea urchin complete.

Development in the sea urchin. Sammichinus miliaris. Third, metamorphosis.

Normal speed, time-lapse, 1 to 96 to 1 to 360. The sea urchin, an inhabitant of the sea bed, has a swimming larva called the echinopluteus. After formation of the axo, hydro, and somato seal, the sea urchin disc is developed through indentation of the body surface in the area of the left hydroseal.

In a larva that is ripe for metamorphosis, the disc almost fills the left part of the body. The disc forms the oral side of the mature sea urchin.

It is supplemented by calcareous structures with pedicellari and spines which lie in the opposite part of the body. With the help of lash bands called epaulettes, the elder pluteus can swim through the water. The metamorphosis from pluteus to sea urchin occurs over a period of about one hour.

It can be initiated by addition of caesium chloride. The movements of the primary podia, now starting, are the first indications of the beginning of metamorphosis.

The sea urchin disc widens into a dome and tears open the amnion skin. The five primary podia now stretch themselves slowly through the opening.

During metamorphosis, cell material from the skeletal rods of the larval arms returns into the body. The melting down of the larval body is shown in quick motion.

The shell-shaped oral side takes up the complete cell material of the larva. Only the calcareous rods of the larval arms, freed of cell material, project out of the body of the larva. The pedicellari, which are shown here on the right of the picture, already possess their full functional ability.

Apart from the outer calcareous rods, no material is lost through metamorphosis. For clarification, the metamorphosis from Pluteus to sea urchin is shown in graphical form.

The elder Pluteus possesses eight larval arms, which are bilaterally symmetrically arrayed and stiffened by rods. These are the dorsal arch, the anterior lateral, post-oral, and posterior dorsal rods. On the left side is the disc, and on the right side are the pedicellari.

The view of the right side of the Pluteus, which is the apporal side of the mature sea urchin, the symmetrical change can be seen well. At the rear end of the dorsal arch, on the right posterior dorsal and post-oral rods, lie three calcareous

networks from which the genital plates 2, 3, and 5 on the apporal side of the sea urchin are later formed. At the beginning of the metamorphosis, the shrinking of the larval body causes these three areas to come closer together.

The cell material of the front part of the larval body is absorbed by the rear part. The genital plates 2, 3, and 5 come into contact with each other. The genital plates 1 and 4 develop from three-pronged formations and grow quickly.

Whilst two young spines develop alongside the pedicellari of the genital plates 3 and 5, plates 1, 2, and 4 develop only one each. The apporal skeleton of the young sea urchin is formed from five almost radially arrayed genital plates and the five terminal plates between them.

Finally, the double rows of thin calcareous plates, which are in ambulacral and interambulacral positions, give the sea urchin skeleton the characteristic radial symmetrical appearance.

During metamorphosis, the internal structure changes also. The sea urchin disc, which is the oral side of the sea urchin, is developed on the left side. During the shrinking of the body, the oral side of the sea urchin enlarges.

Mouth and anus of the larva lose their function. A new mouth forms between the five dental sacs. The young sea urchin, which has just completed its metamorphosis, turns itself on the former left side of the larva.

To the right side of the madripaite plate lies the dorsal sac, which is the remains of the right axocele. Whereas the extensive water vascular system is built by the left hydrocele, the right does not normally appear to have a function.

The right and left somatocele form the coelomatic trunk of the sea urchin. These structural relationships are also retained in the elder sea urchin. On the left side, the proportional relationships of the interambulacral segments are shown, and on the right, those of the ambulacral segments.

The number of plates, podae, pedicillariae and spines have increased. From the five tooth structures, the so-called lamp of Aristoteles is developed, in the centre of which, a new mouth is formed. In the interambulacral area is a tooth, in the ambulacral area, a dental sac.

Opposite the mouth, the anus now appears. Once again, we will show the individual phases of metamorphosis in real life. In the planned view of the oral side of the developing sea urchin, one can recognise melting of the larval arms.

The larval body shrinks to the size of the sea urchin disc. Between the oral and aboral sides, there is a brighter area surrounding the developing sea urchin.

The young sea urchin is still transparent, because the calcareous plates of the skeleton are not yet thickened. The podae are moved by the coelomic liquid pressure of the water vascular system and by their own muscle contractions.

Contractions of the water ring canal change the pressure in the water vascular system.

Calcareous networks surround the newly developing mouth. The five teeth of the sea urchin are formed in dental sacs and they are functional shortly after the metamorphosis.

The increasing calcareous deposit prevents further internal observations.

The calcareous layers are clearly visible in polarised light. The side view shows the structure of the young sea urchin in an upper aboral and a lower oral half. The four pointed young spines mark the position of the genital and terminal plates.

The five pointed radial symmetry of the sea urchin is developed from the bilateral symmetry of the lava by metamorphosis. The sea urchin is now about 2 mm in size and grows continuously by building new skeletal plates.

Development of the sea urchin Sammichinus miliaris, differentiation of sea loam.

The sea urchin develops from a lava, the echinopluteus. The adult is radially symmetrical and moves by ambulacral podia on the bottom of the sea. The planktonic lava, however, has a distinct bilateral symmetry and swims in the water by the movements of its ciliated bands.

Here, in a lateral view, the young pluteus has a complete digestive system with esophagus, stomach, intestine and anus. The lava has an elongated body with four larval arms surrounding the mouth.

Body and arms are stiffened by calcareous skeletal rods. In the dorsal view, only the esophagus and stomach are recognisable. The pluteus is about four days old and is bilaterally symmetrical. This alters during the development of the sea loam, which the following diagram shows.

There are two coelomic sacs, which are formed at the end of the archenteron by division of one coelomic sac. They are seen here to the left and right of the esophagus. The left coelomic sac is larger than the right. This becomes clearer later on in its development.

Both coelomic sacs move back towards the stomach along the plasmatic extensions of the cells of the secondary mesenchyme. Simultaneously, a thin extension grows from the left coelomic sac to the dorsal side of the lava. This is the anlaga of the stone canal.

About seven days after fertilisation, each coelomic sac divides into an anterior and a posterior sac. The anterior parts remain at the esophagus. The posterior sacs move further along mesenchymatic strands towards the stomach.

A little later, the posterior sacs divide again. There thus appears an archimetric articulation of the sea loam in axoseal, hydroseal and somatoseal, known as proseal, mesoseal and metaseal in other invertebrates.

The differentiation of the left coelomic sacs is much greater than that of the right ones. The rudimentary right axoseal is later called the dorsal sac. The right hydroseal disappears and the right somatoseal remains small. Viewed from the left side, the hydroseal is in the central part of the lava.

The axoseal continues beyond the stone canal to the dorsal side of the lava. In this manner, axoseal and hydroseal are connected with the surface of the body. The somatoseal covers nearly the whole left side of the stomach.

From this five days old pluteus, one can see coelomic sacs on both sides of the esophagus. The left one is already larger than the right. On the eighth day, the coelomic sacs divide for the first time.

The anterior parts remain near the esophagus. The posterior parts develop towards the stomach. They divide once again. Now the threefold division of the coelome into axoseal, hydroseal and somatoseal is achieved.

Viewed from the left, one can see the hydropore as a dark spot near the stomach. The hydroporic canal, which connects the left hydroseal with the seawater, ends there. Older larvae are larger and broader. Their arms are also longer.

The lava propels itself with four, here dark-pointed ciliated bands, the epaulettes. At this stage, the development of the sea urchin and lager begins. This is shown in the following diagram. The pluteus has eight larval arms.

The posterior part of the body is rounded. Only two of the four epaulettes are visible. About 14 days after fertilisation, the threefold division into axoseal, hydroseal and somatoseal is finished.

Further development will mainly be in the left hydroseal, which is now barrelled out by a large coelomic cavity. Near the left hydroseal, the epithelium of the body surface will be indented. It thickens and lies together with the central part of the hydroseal.

The outer epithelium of the hydroseal also thickens. During this process, part of the somatoseal pushes slowly between the stomach and the hydroseal. The hydroseal is compressed by the thick ectodermal indentation.

The ectodermal indentation deepens. The hydroseal changes into a peripheral ring canal, the anaga of the ambulacral system.

The edges of the indented surface of the body close together. Beneath is the vestibular cavity. The rounded coelom cavity of the hydroseal has been expanded in five places. Here, the five primary podia develop. In the optical section, two of them are pictured.

They are the first visible organs of the five-pointed radially symmetrical ambulacral system. There are folds of ectoderm, the epineural folds, which run parallel to the radial water canal.

With the aid of the somatoseal, here dark red, the anlagon of the five teeth and the early stages of the hemal system are formed. In this way, the epineural folds arch outwards to form the anlaga of the ectoneurological nervous system, shown here in yellow.

The edges of the epineural folds grow together. The ectoneurological nerve ring develops. On the left side of the pluteus, organ anlagon are formed by indentation or bulging of ectodermal or mesodermal material. From these, the essential parts of the oral side of the sea urchin later develop.

On the right side, which later becomes the ap-oral side of the sea urchin, pedicellari have been formed on the surface of the body. Some stages of differentiation which lead to the radial asymmetrical oral part of the sea urchin are visible in the living pluteus.

This 14-day-old pluteus has a very large left hydroseal with a thick outer coelomic epithelium. The left coelom is rounded off.

The surface of the body is indented and, at the point of contact, the epithelium has thickened. The somatoseal extends and passes under the hydroseal.

The robust epithelium of the ectoderm presses the hydroseal together. The radial water canal develops. The body surface of the larva closes over the vestibule. This gives rise to the embryonic covering or amnion.

The further course of development can be seen better in a lateral view of the larva. We are now looking at the outer surface of the vestibule and can recognize bulges of the hydroseal,

which are the anlagon of the ambulacral system. The bulges, rounding off at the ends of the five radial canals, are the anlagon of the primary podia.

In the centre, the ring canal of the ambulacral system has formed. The five primary podia have developed towards the centre and now cover nearly the whole oral side of the sea urchin.

Under stronger magnification, you can see the first calcareous plates of the developing sea urchin. The sea urchin disc enlarges and the number of calcareous plates increases.

About 30 days after fertilization, the sea urchin disc has reached its greatest diameter. Water currents are observable in the ring canal of the ambulacral system. They are responsible for the first movements.

During differentiation of the sea urchin oral side to the left of the larval stomach, pedicillariae are formed on the surface of the body to the right. Viewed from above, each pedicillariae is seen to have three valves.

With older plutei, spines are also visible beside the pedicillariae. About five weeks after fertilization,

the pluteus of Sammichinus miliaris is ready for metamorphosis.