Full Lecture Notes on Embryology for Medical Students!
Embryology Note # 1. Fate Maps of an Early Embryo:
A correct interpretation of gastrulation is impossible without a knowledge of the position which the presumptive germinal layers occupy in the blastula. This position may be ascertained in various ways. A chart, showing the fate of each part of an early embryo, in particular, a blastula, is called a fate map.
In tracing the fate of various parts of the blastoderm, it is sometimes possible to make use of the peculiarities of the cytoplasm in certain parts of the egg, such as the presence of pigment granules. In the developing amphibian egg, for instance, one may trace in which part of the differentiated embryo the black pigment comes to lie.
Originally this pigment is restricted to the animal hemisphere of the egg. However, peculiarities of pigmentation are seldom sufficient to make it possible to reconstruct the fate map in any great detail. Recourse must be had to artificial marking of parts of the blastoderm.
A satisfactory method of marking was devised for this purpose by Vogt (1925). The method consists in soaking a piece of agar in a vital dye (Nile blue sulfate, neutral red, Bismarck brown) and then applying the piece of agar to the surface of the embryo in the necessary position.
The dye diffuses from the agar, and in a matter of minutes the cells of the embryo to which the agar has been applied take up sufficient dye to produce a stain on the surface of the embryo. This marking-can is done without removing the vitelline membrane, since it is permeable to the vital dyes, and thus the embryo continues to develop normally.
The presence of the stain does not change the normal development of the embryo, and the position of the stained cells in the differentiated embryo clearly shows the fate of the stained area. It has been established by trial that the vital dye remains, on the whole, restricted to the cells which had originally taken up the dye and to their descendants.
The diffusion of the stain, if the staining has been done correctly, is negligible and does not interfere with interpreting the results. Several stain marks may be made on the surface of the same embryo, using different colors (red, blue, brown). In this way one experiment may disclose the fate of many parts of the early embryo at the same time.
It was later found that cellophane can also be used as a stain carrier instead of agar and that it is actually a more convenient one, as cellophane comes in thin sheets from which it is easy to cut out pieces of the desired size and shape. All vital staining is now done with cellophane as the stain carrier.
Independently of vital staining, another method has also been devised for marking cells of a developing embryo. This consists in applying tiny particles of carbon to the 196 surface of the embryo. Carbon particles stick to the surface of the cells and can thus be used as markers enabling the investigator to follow the movements of the cells and to draw up fate maps.
The vital stain marking method was first applied to the reconstruction of the fate map in the amphibian embryo, and the original investigations have subsequently been checked by many embryologists. It is most advantageous, therefore, that the fate map of an amphibian embryo, such as the embryo of a newt (Triturus) or axolotl (Ambystoma), be described first.
The Fate Map of a Urodele Amphibian Embryo:
The whole surface of the blastula of an amphibian may be roughly divided into three main regions:
(1) A large area on and around the animal pole,
(2) An intermediate zone, also known as the marginal zone, extending all around the equator of the blastula, and
(3) The area on and around the vegetal pole.
These three main regions coincide approximately with areas which differ in their pigmentation. The whole of the animal region is deeply pigmented. The marginal zone, which is much broader on one side of the embryo, is pigmented but not so deeply as the region around the animal pole. The vegetal region has very little pigment.
Inside each region we find areas corresponding to the future organs of the animal. The animal region consists of two main areas: the area whose fate is to develop into the nervous system of the embryo, and the area which is to become the skin epidermis of the embryo. The material for the sense organs is also contained in these two areas.
Inside the nervous system area, a small subarea may be traced which is to participate in the formation of the eyes of the embryo; inside the epidermis area, the material for the nose, the ears, and the ectodermal part of the mouth may similarly be traced. In the intermediate or marginal zone, we find the material for the notochord occupying a large area on the dorsal side of the blastula.
This is followed by an area lying nearer to the vegetal pole and containing the material for the prechordal connective tissue; this area is known as the prechordal plate. Farther down, toward the vegetal pole but still inside the marginal zone, lies the material for the anterior parts of the alimentary canal – the endodermal lining of the mouth, gill region, and pharynx.
The parts of the marginal zone on both sides of the notochordal area are taken up by the material for the segmental muscles of the body. The lateral and ventral parts of the marginal zone give rise to the mesodermal lining of the body cavity, the kidneys, etc. (ventrolateral mesoderm). The vegetal region is composed of cells which are later found in the midgut and hindgut.
Local vital staining of the surface of the embryo, if performed carefully, leaves the color only in the superficial cells; strictly speaking, the fate map shows only the destination of the cells of the superficial layer.
The blastoderm of the amphibian embryo in the blastula stage, however, is not a simple (that is, single-layered) epithelium but a stratified epithelium, and the nearer it is to the vegetal pole, the greater the number of layers of cells. Yolk-laden cells are stacked in large numbers on top of one another in the vegetal hemisphere of the blastula.
The number of cell layers is least at the animal pole, but in the marginal zone the blastoderm is many cells thick. The cells lying in the interior in the marginal zone are intermediate in size and yolk content between the cells of the vegetal field and the cells of the animal hemisphere, and form a ring in the interior of the blastula, called the inner marginal zone.
The destiny of the cells not reaching the surface need not be the same as those on the exterior, and their fate cannot be ascertained from surface color marks. In fact, Vogt could not detect the location in the blastula of the material for some mesodermal structures: the cells going into the formation of the heart, and of the blood islands. He concluded that these parts develop from cells of the inner marginal zone.
Vogt, using the same methods, also constructed a fate map for a frog blastula, that of Bombina. The general arrangement of the various areas is the same as in the fate map for a urodelean blastula, but all presumptive areas are more concentrated toward the marginal zone. In particular, the area for the neural plate does not reach, by far, the animal pole.
A fate map has been worked out also for an earlier stage of an amphibian egg, namely for the 32-blastomere stage of the frog Xenopus laeuis. At the 32-cell stage the blastomeres are arranged in four tiers, each tier consisting of eight cells. For reference the blastomeres belonging to each tier are designated by capital letters – A for the topmost tier (the tier at the animal pole), B, C, and D for the following tiers in descending order.
The tier C forms the marginal zone, and the tier D consists of the vegetal pole blastmeres. Within each tier the blastomeres are designated by the numbers 1-4, on each side, starting from the mid-dorsal plane. Each individual blastomere was stained with Nile blue sulfate, following Vogt’s method. The essential results of the staining were as follows.
Blastomeres A1, right and left, give rise to the anterior end of the neural plate, including the eye rudiments, and the transverse neural fold. The rest of the neural plate is contained in the blastomeres B1 B2 and C2. The epidermis is produced from blastomeres A2 A3 A4 B2 B3 B4 and C3 C4. Most of the notochord is derived from the blastomeres B1, right and left. The posterior part of the notochord is derived from blastomeres C1, right and left.
The same blastomeres also contain the material for the foregut. Most of the somites are derived from the blastomeres C2 and C3. The blastomeres of the D tier go entirely into the formation of the gut, the blastomeres D1 D2 and D3 forming the midgut, and the blastomeres D4 forming the hindgut. Blastomeres C3 and C4 also contribute to a slight extent to the formation of mid- and hindgut.
Comparing the fate map arising from this experiment with the fate map for a frog blastula (Fig. 128), two essential differences may be noted. Firstly, the neural plate area reaches further toward the animal pole in the morula stage; in fact, the transverse neural fold lies at the animal pole. This shows that between cleavage stages and the beginning of gastrulation there is a shifting downward of the presumptive neural material, toward the equator.
This shifting was actually noted by Nakamura and Kishiyama. The second difference concerns a much greater concentration of the material for the notochord toward the mid-dorsal plane. It is thus possible that between cleavage and the beginning of gastrulation, while the presumptive neural material shifts downward toward the equator, the equatorial material, the presumptive notochord in particular, spreads out sideways.
In the 32-cell stage all blastomeres lie superficially, and are presumably stained right through. There are no blastomeres in the interior. Thus parts of the cells which will later become internal, in particular cells of the inner marginal zone, would all be stained in the present experiment. Rudiments later located in the superficial layer, and those derived later from cells lying in the interior, May well be derived from the same blastomeres of the 32-cell embryo.
It will be noticed that the areas destined to develop into the organs of the mid-dorsal part of the animal lie on one side of the blastula, on that side where the marginal zone is taken up by the notochord area. Nearer to the animal pole the area of the neural system is situated. This side of the blastula corresponds therefore to the dorsal side of the embryo.
Similarly, parts of the head (eye, nose, ears) develop from areas near the animal pole of the blastula, which therefore corresponds to the anterior end of the embryo. Since the materials of the egg have not been displaced to any great extent during the cleavage, it may be inferred that the animal pole of the fertilized egg corresponds to the anterior end of the embryo.
The side where the marginal zone is the broadest is the dorsal side; the opposite side may be considered as ventral, and the vegetal pole as the posterior. We find, however, that the foregut area (pharynx, part of the epithelium of the mouth) is also situated on what we have agreed to call the dorsal side of the egg.
On the whole, however, the location of the areas destined to develop into most of the organs does not seem to have anything in common with the position of the same organs in the adult animal.
Organs which later are situated in the interior of the animal’s body—such as the notochord, the gut, or the brain—are represented by areas laid out on the surface of the blastula. The cells destined to cover the whole of the animal’s body, such as the epidermis of the skin, occupy only a limited area on the surface of the blastula.
Parts of the three germinal layers—ectoderm, endoderm, and mesoderm—are all located on the surface of the embryo, instead of being in a concentric arrangement, with the ectoderm on the outside, the endoderm in the middle, and the mesoderm in between. It is obvious that a far-reaching displacement or reshuffling of the parts of the blastoderm must take place before each cell can arrive at its final position.
This displacement of parts of the blastoderm, which are eventually rearranged in a system of concentric layers of cells, is the essence of the process of gastrulation. We shall now, for a time, leave the amphibians and consider the process of gastrulation in an animal with a more simple organization, a representative of lower chordates, the Amphioxus.
Embryology Note # 2. Translation in Eukaryotes:
There is a fundamental difference in the processes that lead to the synthesis of proteins as directed by the mRNA in prokaryotes, compared with eukaryotes. In prokaryotes the mRNA becomes associated with ribosomes immediately on its formation, and some parts of the mRNA molecule start being “read”—directing the assembly of amino acids into a polypeptide, while the remaining parts of the mRNA molecule are still in the process of being polymerized on the DNA template.
In eukaryotes the protein synthesis takes place in the cytoplasm, after the mRNA has passed out of the nucleus, and becomes associated with the cytoplasmic ribosomes. It is the separation in time and space of the transcription and translation that provides the opportunity for the molecules of the hnRNA to be processed and transformed into the functional mRNA.
The ribosomes of prokaryotes and eukaryotes also have a different structure – prokaryotic ribosomes are smaller and have the sedimentation constant 70S; eukaryotic ribosomes are larger and, with a few exceptions, have a sedimentation constant of 80S.
Ribosomes of eukaryotes are either free in the cytoplasm or are attached to membranes of the endoplasmic reticulum. In the latter case proteins Synthesized in the ribosomes become inserted into the membrane of the endoplasmic reticulum, or they are pushed into the cavity of the reticulum while the rear end of the molecule is still in the process of being assembled.
The forward part of the protein molecule, serving to insert the molecule into the membrane, or to draw it into the cavity of the reticulum, is rejected after the penetration has been achieved.
This part of the polypeptide molecule is called the “signal sequence,” and is about 30 amino-acid residues long. Accordingly, the DNA sequence coding for this section of the polypeptide (± 90 nucleotides long), and the corresponding part of the mRNA molecule, have no representation in the finally produced protein.
The rate of protein synthesis in eukaryotes is lower than in prokaryotes. In bacteria about 20 amino-acid residues are added to the polypeptide chain in one ribosome per minute, while in mammalian ribosomes only about one amino-acid residue is added in a ribosome in the same time.
Embryology Note # 3. Synthesis of DNA-DNA and DNA-RNA Duplexes:
The study of genetic information carriers—DNA and RNA—depends to a large degree on the possibility of identifying sequences of these materials; that is, determining which sequences are similar or identical, and which are dissimilar. A complete chemical analysis of each molecule would be the most satisfactory method, but such an analysis, although possible, is very laborious, and it needs a large amount of identical material and thus is not suitable as a first approach to the goal.
Fortunately another much simpler, and very efficient, method has been discovered—the method of molecular hybridization. In the first instance this method leads not to the recognition of similar (or identical) sequences, but to the recognition of complementary sequences. In the double-stranded molecule of DNA the two strands are held together because the bases in each strand have a corresponding, complementary base in the other strand – adenine being complementary to thymidine, and guanine being complementary to cytosine.
If the two strands were to be separated and then brought together, they would reunite by each base finding a complementary base in the other strand. If, instead of bringing together the two strands originally separated from each other, one of the strands is associated with a strand of different origin but containing the complementary bases in the same order as in the original strand, such strands could unite. Furthermore, any strand that can unite with a given strand would be proved to be identical to any other strand that has the same capacity for joining the original or “test” strand.
The separation of two strands of DNA can be achieved by heating a solution containing DNA to above 65-70° C. On cooling the solution below this temperature, strands of DNA start joining together if they contain complementary sequences of nucleotides, in a process called renaturation.
Molecules of RNA may have sequences of nucleotides which are complementary to the sequences in the DNA as, for instance, in the case of RNA synthesized on a DNA template (except that a uridine nucleotide in the RNA is complementary to the adenine nucleotide in the DNA). Thus, DNA-RNA duplexes may be produced by this method.
If molecules of DNA and RNA join together, the term molecular hybridization is used for this process. Again, there is here a possibility of recognizing similar sequences both in DNA and RNA; all DNA sequences which hybridize with the same RNA sequence must be recognized as identical or at least similar, and all RNA sequences which hybridize with the same DNA sequence must be identical or similar among themselves.
Once the complementary strands have become associated, these can be separated from the single strands (the ones that for one reason or another did not find a suitable partner) by one of two methods. The first method is to use an enzyme, a nuclease, which specifically degrades single-stranded DNA (the S1 nuclease, obtained from Aspergillus oryzae).
All single-stranded DNA molecules become destroyed, and only the double- stranded fragments remain intact. In the second method the property of hydroxyapatite (calcium phosphate) of binding double-stranded but not single-stranded DNA is exploited.
The DNA fragments in 0.12 M sodium phosphate buffer at pH 6.8 are passed through a column containing hydroxyapatite which binds the duplexes containing more than 50 base pairs, whereas single-stranded material is not absorbed, and passes through. The duplexes are then eluted (washed out) with 4 M phosphate buffer.
The amount of re-association of DNA or DNA/RNA molecules depends mainly on two factors. Firstly, it depends on the concentration of the two complementary sequences—the more molecules that are in solution, the greater the chances for two complementary molecules to meet, an obvious prerequisite for their joining together.
Secondly, the reaction depends on the time during which the reaction is allowed to proceed—the longer the time, the greater the number of complementary molecules that have a chance to collide and join together. These two factors are reflected in a parameter which has been introduced to characterize the dynamics of the re-association of nucleic acid molecules – the Cot, which is simply the product of concentration of the nucleotides in moles per liter by time in seconds.
The process of re-association can be represented by plotting the Cot on the abscissa, and the proportion of the nucleotides that have formed duplexes on the ordinate. As the value of Cot varies over several orders of magnitude, these values are most conveniently plotted on a logarithmic scale.
The result of such plotting is, in the case of a homogeneous population of nucleotide sequences (that is, when all the sequences in the genome are present in only a single copy), a simple sigmoid curve. In interpreting the curve it must be borne in mind that the curve does not represent the progress of the reaction in time, as the same value for the amount of re-association may be reached, owing to either a greater initial concentration or the reaction having been run for a longer time.
In the case of heterogeneous populations of polynucleotides, which are present in different concentrations the curve is not simple; different groups of molecules associate at different Cot values, and correspondingly the curve may have several rising sections.
The knowledge of the dynamics of molecular hybridization allows the use of this method for determining some quantitative characteristics of certain DNA and RNA sequences. A rare sequence that is present only in low concentrations in any sample will need a much longer time to form duplexes because the chances of such molecules colliding with a complementary partner are small; a sequence that is in great abundance in a sample will re-associate rapidly, because the chances are greater that such molecules will collide with a suitable partner.
Thus the hybridization experiment can give an estimate of the numbers of certain types of molecule in a sample. Furthermore, the method can be used for separation in a mixture of the sequences that are abundant, or common, from the sequences that are rare. It is only necessary to run the reaction for a sufficient time for the common sequences to associate, and then separate them from the molecules remaining single on a hydroxyapatite column.
It was discovered through the use of the molecular hybridization method that in the genomes of eukaryotes some sequences of the DNA are present in multiple copies. As it is not possible at this time to separate particular sections of the DNA performing different functions (sections representing specific genes, or other functional units), recourse has been made to breaking up the DNA from the chromosomes randomly into fragments.
This is done either by using ultrasound, or by squirting a solution of DNA under high pressure through a narrow orifice, or by high-speed blending. The chromosomal DNA is thus broken up into fragments about 500 nucleotides in length. These are then dissociated into a single-stranded condition, and allowed to re-associate.
It is found that some fragments associate very rapidly, showing that they all contain similar (complementary) sequences of nucleotides, while other fragments take a very long time to re-associate, showing that the nucleotide sequences in these fragments were not repeated in the same genome.
Embryology Note # 4. Envelopes of the Egg- Explain
All eggs, like any other cells, are of course covered by the cell membrane or plasmalemma. Under the electron microscope the plasmalemma sometimes can be seen to be double, with two layers of electron-dense material about 50 Å thick separated by a less dense interval of about 60 Å.
In addition to the plasmalemma, eggs of all animals, with the exception of sponges and some coelenterates, are surrounded by special egg envelopes. Depending on their origin these may be subdivided in two groups – the primary envelopes and the secondary envelopes (In older literature, the egg envelopes were referred to as egg membranes).
The primary egg envelopes are those which develop in the ovary between the oocytes and the follicle cells in the space occupied by the inter-digitating microvilli. In the initial stages of its formation the envelope shows positive reactions for mucopolysaccharides, but in later stages it is fortified by addition of further substances and may consist of fibrous proteins, or in insects, it may be sclerotized and may become highly impermeable.
The envelope thus formed bears different names in different animals – it is known as the vitaelline envelope or vitelline membrane in insects, molluscs, amphibians, and birds; in tunicates and fishes it is usually called the chorion. In mammals the envelope of an exactly similar nature is called the zona pellucida. The zona pellucida thus takes the place of the zona radiata. The jelly coat surrounding the eggs of sea urchins belongs in the same group.
The origin of the materials forming these envelopes is not quite obvious; usually the envelope is believed to be secreted by the follicle cells, but the oocyte may perhaps contribute to the formation of the envelope in some cases.
In insects a second, thicker coat is secreted by the follicle cells on top of the vitelline envelope. This second envelope is called the chorion. In many insects its surface shows a complicated sculpture, which is typical for each species. The primary egg envelopes usually closely adhere to the surface of the oocyte, but at a later stage a space filled with fluid may appear between the cytoplasm and the envelope; this space is called the perivitelline space.
In mammals an egg escaping from the ovary carries with it on the surface of the zona pellucida a layer of follicle cells known as the corona radiata. The cells of the corona radiata are peeled off later, as the egg descends the oviduct.
The second group of egg envelopes is those which are secreted by oviducts and other accessory parts of the genital organs while the egg is passing from the ovary to the exterior.
The eggs of amphibians are surrounded by a layer of jelly, which protects the egg and sometimes serves to make the eggs adhere to one another and to submerged objects such as water plants. This jelly is secreted as the eggs pass through the oviducts.
When the amphibian egg is deposited in water, the jelly absorbs water and swells. In the oviparous sharks and rays the egg is surrounded in the oviducts (in the special parts called the shell glands) by a hard shell of a complicated shape. The shell is drawn out into long twisted horns which serve to entangle the eggs among seaweed.
The most complicated egg envelopes, however, are found in the eggs of birds, where no less than five envelopes can be distinguished, the innermost being the vitelline envelope—a very thin envelope covering the surface of the yellow of the egg (which is the true egg cell). This envelope actually has a double origin.
An inner layer of the envelope is produced in the ovary, in the space between the oocyte and the follicle cells. This layer is composed of very rough fibers. An outer, more finely fibrous layer is then formed on top of the first layer when the egg enters the upper portion of the fallopian tube.
The next envelope is the white of the egg. Eighty-five per cent of the egg white is water; the rest is a mixture of several proteins, mostly albumins, which make up 94 per cent of the dry weight. A denser part of the egg white forms strands, known as the chalazae, which help to keep the egg cell in the center of the egg white. Next to the egg white are two layers of shell envelopes which consist of fibers of keratin matted together.
Over most of the surface of the egg the shell envelopes are in contact with one another, but at the blunt end of the egg they are separated; the inner envelope adheres to the egg white, and the outer envelope adheres to the shell, leaving a space in between filled with air. The outermost envelope is the shell, which consists chiefly of calcium carbonate (CaCO3), about 5 gm. in a hen’s egg.
The shell is pierced by many fine pores which are filled by an organic (protein) substance related to collagen. In an average hen’s egg the pores have a diameter of 0.04 to 0.05 mm., and the total number of pores is estimated at about 7000. The envelopes of a bird’s egg are secreted one after another as the egg proceeds down the oviduct. The whole process takes slightly longer than 24 hours.
After the egg has been released from the ovary, it quickly passes into the oviduct and descends through it for about three hours, during which most of the egg white is secreted and envelops the egg cell. The lowest portion of the oviduct is widened and is termed the uterus. Here the egg remains for 20 to 24 hours, while the remainder of the egg white and eventually the shell envelopes and the shell itself are secreted.
Not only do the envelopes of a bird’s egg protect the egg cell, but the egg white serves also as an additional source of nourishment and is gradually used up in the course of the development of the embryo.
Embryology Note # 5. Components of Spermatozoon:
There is some variation in different animals as to how much of the spermatozoon is taken into the interior of the egg. In many animals, notably in the mammals, all the internal parts of the spermatozoon, nucleus, and components of the middle piece and the axial filaments of the tail penetrate into the cytoplasm and for a short time may be seen lying intact in the interior of the egg.
In some animals (echinoderms), however, the tail of the spermatozoon breaks off and is left outside the vitelline membrane, and even the middle piece of the spermatozoon may be left without, so that only the nucleus and the centrosome enter the egg (Nereis). That the tail of the spermatozoon often does not enter the egg gives additional proof that its functions are purely locomotive.
The information concerning the middle piece of the spermatozoon is not unequivocal. Although components of the middle piece appear to enter the egg cytoplasm in most cases, there is no definite proof that any constituents of the spermatozoon except for the nucleus and the centrosome play an active part in subsequent development. The mitochondria contained in the middle piece have been observed in some cases to scatter in the cytoplasm of the egg, but it is not known how long, they maintain their existence there.
The subsequent behavior of the spermatozoon nucleus is dependent on the stage of maturation (reduction divisions) which the egg has reached at the time of fertilization. In the sea urchins the eggs are shed and become fertilized after the reduction divisions have been completed and both polar bodies have been extruded from the egg. This is, however, by no means a general occurrence.
In vertebrates the rule is that the egg completes its first reduction division in the ovary and reaches the metaphase stage of the second meiotic division. At this stage all further progress is arrested, ovulation takes place, and the egg may become fertilized. The second reduction division is completed and the second polar body extruded only if the egg is fertilized by a spermatozoon or activated in some other way.
In ascidians, some molluscs, and annelids, the egg reaches only the metaphase of the first meiotic division when it becomes ripe and is fertilized; only then does the egg complete the first reduction division and carry out the second. Lastly, in some annelids, in nematodes, and in chaetognaths, the eggs are fertilized even before the beginning of meiotic division, while the oocyte nucleus is still intact.
It follows that although in animals like the sea urchins the spermatozoon nucleus may immediately proceed to join the egg nucleus, in other cases the immediate effect of the fertilization, as far as the nuclear apparatus is concerned, is the completion of the meiotic divisions, only after which the fusion of the male and female pronuclei may take place.
When the spermatozoon first fuses with the egg cytoplasm, it moves with the acrosome (or acrosomal filament) at its front. The nucleus and the centrosome, in that order, are arrayed behind the acrosome. After fusion of the two gametes, however, a rotation of the nucleus and the centrosome can be observed in many, though not in all, animals, the centrosome coming ahead of the nucleus and the nucleus turning 180 degrees so that its original posterior end turns forward.
The other parts of the spermatozoon, if still discernible by this stage, lose connection with the nucleus and the centrosome. Both the nucleus and the centrosome change in appearance. The nucleus, which is now referred to as the male pronucleus, starts swelling, and the chromatin, which is very closely packed in the spermatozoon, again becomes finely granular.
By imbibition of fluid from the surrounding cytoplasm the pronucleus becomes vesicular. The centrosome, at the same time, becomes surrounded by an aster, similar to that of the centrosome in the early stages of an ordinary mitosis. While these changes are occurring, the sperm nucleus, together with the centrosome, moves through the egg cytoplasm toward the area where fusion with the egg nucleus, the female pronucleus, is to take place.
This area is generally near the center in holoblastic eggs having a fairly small amount of yolk, but in telolecithal eggs it is in the center of the active cytoplasm at the animal pole of the egg. As the sperm head moves inward it may be accompanied by some cortical and subcortical cytoplasm. If the latter is heavily pigmented, as in amphibian eggs, the trajectory of the sperm head may be marked by pigment granules trailing along its path. This is sometimes referred to as the penetration path.
The female pronucleus also has to traverse a greater or lesser way before it reaches the male pronucleus. At the beginning of its migration the female pronucleus is invariably at the surface of the egg, where the second meiotic division has been taking place, since it is only after the completion of the meiotic division that the nucleus of the egg may fuse with the nucleus of the spermatozoon.
The haploid nucleus of the egg, after the completion of the second meiotic division, is often in the form of several vesicles known as the karyomeres. This fuse together to form the female pronucleus, which swells and increases in volume as it approaches the male pronucleus. In the last stage before they meet, the male and female pronuclei may become indistinguishable.
The actual fusion of the male and female pronuclei to form a single zygote nucleus may differ in detail among different animals. In some animals the two pronuclei actually fuse together; that is, the nuclear membranes become broken at the point of contact, and the contents of the nuclei unite into one mass surrounded by a common nuclear membrane – the zygote nucleus.
At the approach of the first cleavage of fertilized eggs of sea urchins and vertebrates, the nuclear membrane dissolves, and the chromosomes of maternal and paternal origin become arranged on the equator of the achromatic spindle. In other cases, however, the male and female pronuclei do not fuse as such, but the nuclear membranes in both dissolve and the chromosomes become released.
In the meantime, the centrosome of the spermatozoon has divided in two and a spindle has been formed to which the chromosomes derived from the male and the female pronuclei become attached. (It is important to note that in normal fertilization the achromatic figure of the first and subsequent cell divisions is produced by the centrosome off the spermatozoon.)
Only after completion of the first division of the fertilized egg do the paternal and maternal chromosomes become enclosed by common nuclear membranes in the nuclei of the two daughter cells into which the egg has become divided (Ascaris, some molluscs, and annelids). In both types of fusion the chromosomes of the maternal and paternal sets retain, of course, their individuality.
Lastly, in some animals, of which the copepod Cyclops is a well-known example, the paternal and maternal nuclear components remain separate for some time, even after cleavage has started, so that each blastomere has a double nucleus consisting of two parts lying side by side, but each surrounded by its own nuclear membrane.
A closer union of the homologous chromosomes takes place much later in preparation for meiosis in the gonads of the new individual and also in cases of somatic conjugation of chromosomes, as in the salivary gland chromosomes of Drosophila.
Embryology Note # 6. Compare between Morula and Blastula:
The blastomeres in the early cleavage stages tend to assume a spherical shape like that of the egg before cleavage. Their mutual pressure flattens the surfaces of the blastomeres in contact with one another, but the free surfaces of each blastomere remain spherical, unless these outer surfaces are also compressed by the vitelline membrane. The whole embryo acquires, in this stage, a characteristic appearance resembling a mulberry. Because of this superficial similarity, the embryo in this stage is called a morula (Latin for mulberry).
The arrangement of the blastomeres in the morula stage may vary in the different groups of the animal kingdom. In coelenterates it is often a massive structure, with blastomeres filling all the space that had been occupied by the un-cleaved egg. Some of the blastomeres then lie externally and others in the interior. (Some embryologists apply the name morula to this type of embryo only.)
More often, as the egg undergoes cleavage, the blastomeres become arranged in one layer, so that all the blastomeres participate in the external surface of the embryo. In this case a cavity soon appears which at first may be represented just by narrow crevices between the blastomeres, but which gradually increases as the cleavage goes on. This cavity is called the blastocoele.
As cleavage proceeds, the adhesion of the blastomeres to one another increases, and they arrange themselves into a true epithelium. In cases in which a cavity has been forming in the interior of the embryo, the epithelial layer completely encloses this cavity, and the embryo becomes a hollow sphere, the walls of which consist of an epithelial layer of cells. Such an embryo is called a blastula. The layer of cells is called the blastoderm, and the cavity is the blastocoele.
Right from the beginning of cleavage the blastomeres become progressively joined closer together by several types of intercellular junctions. The first to be formed are the “tight” junctions forming at the outer edges of the adjoining blastomeres, sometimes already in the two-blastomere stage. At the blastula stage these junctions seal off the interior of the embryo from the outside.
In the sea urchin blastula these junctions take the form of “septate” junctions, which appear as a series of bars connecting the membranes of adjoining cells. “Gap” junctions which provide a means of communication between cells of the embryo are absent in the earliest stages, but develop in the morula stage and are widespread in the blastula stage.
In oligolecithal eggs with complete cleavage (echinoderms, Amphioxus), the blastomeres at the end of cleavage are not of exactly equal size, the blastomeres near the vegetal pole being slightly larger than those on the animal pole. When the blastula is formed, the cells arrange themselves into a simple columnar epithelium enclosing the blastocoele.
Because the vegetal cells are larger than the animal cells, the blastoderm is not of an equal thickness throughout; at the vegetal pole the epithelium is thicker, and at the animal pole it is thinner. Thus, the polarity of the egg persists in the polarity of the blastula.
Animals with a larger amount of yolk, such as the frog, show a difference in the size of the cells of the blastula that may be very considerable, and the blastula still further departs from the simple form of a hollow sphere. The cells here are also arranged in a layer surrounding the cavity in the interior, but the layer is of very uneven thickness.
The layer of cells at the vegetal pole is very much thicker than at the animal pole, and the blastocoele is consequently distinctly eccentric, nearer to the animal pole of the embryo (Fig. 94b). Furthermore, the blastoderm is no longer a simple columnar epithelium but is two or more cells thick.
The cells in the interior are rather loosely connected to one another, but at the external surface of the blastula the cells adhere to one another very firmly, because of the presence of tight cell junctions joining the surface of adjoining cells in a narrow zone just underneath the surface of the blastoderm.
A process corresponding to blastula formation occurs also in animals whose eggs have incomplete cleavage. In a bony fish or a shark the early blastomeres tend to round themselves off, showing that they are only loosely bound together. Later, the blastomeres adhere to one another more firmly and thus become converted into an epithelium.
Again, as in amphibians, the superficial cells are firmly joined to one another, forming a continuous “covering layer,” while the cells in the interior may remain loosely connected until a later stage. The epithelium, however, cannot have the form of a sphere.
Since the cleavage is restricted to the cap of cytoplasm on the animal pole of the egg, the blastoderm is developed only in the same polar region. The blastoderm therefore assumes the shape of a disc lying on the animal pole. The disc, which is called the blastodisc, is more or less convex and encloses, between itself and the un-cleaved residue of the egg, a cavity representing the blastocoele.
In centrolecithal eggs having a superficial cleavage (insects), there is no cavity comparable to the blastocoele. Nevertheless, the formation of the epithelium on the surface of the embryo, after the nuclei have migrated to the exterior, can be compared to the formation of the blastula.
The layer of cells thus formed on the surface of the embryo is the blastoderm. Instead of surrounding a cavity, the blastoderm envelops the mass of un-cleaved yolk. We may also compare this stage to an embryo whose blastocoele has been filled with yolk.
Up to the blastula stage, the developing embryo preserves the same general shape as the un-cleaved egg. So far, the results achieved are the subdivision of the single cell into a multiplicity of cells and the formation of the blastocoele. In addition, the substances contained in the egg remain basically in the same position as before.
The yolk remains near the vegetal pole. In pigmented eggs, such as those of amphibians, the pigment remains as before, more or less restricted to the upper hemisphere of the embryo. Only a slight intermingling of cytoplasm seems to be produced by the cleavage furrows cutting through the substance of the egg.
We have pointed out that during cleavage qualitative changes in the chemical composition of the developing embryo are very limited. Few new substances, either chemically defined or microscopically detectable, have been found to appear during cleavage. It is conceivable, however, that the substances present in the egg may be redistributed in some way during cleavage and that such a redistribution may be essentially important for further development.
Embryology Note # 7. Gastrulation in Amphioxus:
In Amphioxus, there are differences in the various regions of the egg cytoplasm which permitted Conklin (1932) to trace these regions into the later stages of development and thus to reconstruct a fate map, at least in rough outlines.
At the beginning of cleavage three regions can be distinguished in the Amphioxus egg. Near the vegetal pole a mass of cytoplasm is found which contains the greatest amount of yolk (although yolk in this case is not abundant and the yolk granules are relatively very small). The animal hemisphere of the egg consists of cytoplasm that has less yolk and is consequently more transparent.
On one side of the egg, in a position roughly corresponding to the marginal zone of the amphibian egg, there is a special type of cytoplasm; it does not contain much yolk, but it can be distinguished from the animal cytoplasm by its ability to be deeply stained by basic dyes. The mass of cytoplasm of this kind has a crescentic shape, the attenuated ends of the crescent being drawn out along the equator of the egg about halfway around.
During the period of cleavage the three regions become subdivided into blastomeres without the cytoplasmic substances having been displaced to any great extent. The distinctions which could be traced in the cytoplasm of the egg now become accentuated by further distinctions in the size and shape of the blastomeres.
The vegetal material is now contained in a number of rather large cells taking up the position on and around the vegetal pole of the blastula. The animal hemisphere is made up of cells containing the clear cytoplasm. The cells are columnar and form a very closely packed columnar epithelium.
The cells containing the basophilic cytoplasm are clearly discernible even as to shape. They are the smallest cells in the blastula, even smaller than the animal cells, and they are rather loosely packed, the external surfaces bulging out, as is usually found in the earlier cleavage stages.
The fate of the three regions is the following: The clear cytoplasm that later becomes the animal hemisphere of the blastula develops mainly into skin epidermis. The granular cytoplasm, which takes up the region around the vegetal pole of the blastula, develops into the lining of the alimentary canal. The crescent of basophilic cytoplasm is the material which gives rise to the muscles and the lining of the body cavity and thus represents a mesodermal area.
More recently, the method of local vital staining has been applied to the study of Amphioxus development. It was found that the presumptive mesodermal area is not restricted to one half of the egg only but reaches farther around the equator.
On the opposite side a zone giving rise to the notochord could be detected, and above that a crescentic area which develops into the nervous system. The similarity between this fate map and that of the amphibians is practically complete. There is also a striking similarity to the distribution of different kinds of cytoplasm in the ascidian embryo.
As a result of cleavage in Amphioxus, a ‘blastula is formed which has a large blastocoele and a blastoderm consisting of a single layer of columnar cells. The cells at the vegetal pole are somewhat larger than at the animal pole, and the blastoderm therefore is thicker.
Gastrulation is initiated when the blastoderm at the vegetal pole becomes flat and subsequently bends inward, so that the whole embryo, instead of being spherical, becomes converted into a cup-shaped structure with a large cavity in open communication with the exterior on the side that was originally the vegetal pole of the embryo.
The cup has a double wall, an external one and an internal one, the latter lining the newly formed cavity. The external and internal epithelial layers are continuous with each other over the rim of the cup-shaped embryo. In this stage there is still a space between the external and internal walls representing the remnants of what was the blastocoele of the blastula.
The external lining consists of presumptive epidermis and presumptive nervous system. In other words, it consists of parts which have been classified as ectoderm. The internal lining consists mainly of the presumptive gut material, that is, of endoderm.
The presumptive material of the notochord and the-mesodermal crescent at first lie on the rim of the cup but very soon they shift inward so as to occupy a position on the internal wall of the cup. In this way the endoderm, the mesoderm, and the notochord disappear from the surface of the embryo into the interior where they belong. The external surface of the embryo now consists of ectoderm.
The embryo in this stage of development is called a gastrula. The movements of infolding or inward bending of the endoderm and mesoderm are known as invagination. The cavity arising through the invagination of the endoderm and mesoderm is called the primary gut or archenteron. The opening of the archenteron to the exterior is called the blastopore.
At the same time, the blastopore denotes the pathway by which the endoderm and mesoderm pass into the interior of the embryo. The blastopore, being the opening leading into the primary gut, has been likened to a mouth; its rims, therefore, are usually referred to as the lips of the blastopore. We may distinguish the dorsal lip, the ventral lip, and the lateral lips of the blastopore, respectively.
The blastopore is very broad in the initial stage of gastrulation, but soon the lips of the blastopore begin to contract, so that the opening which leads into the archenteron becomes smaller and is eventually reduced to an insignificant fraction of the original orifice.
This contraction of the lips of the blastopore is connected with the disappearance of the mesodermal crescent material and the presumptive notochord from the rim of the cup-shaped embryo. As more material is shifted into the interior of the gastrula, the remnants of the blastocoele become completely obliterated by the two walls of the embryo coming in contact with each other.
As the presumptive notochord and the mesodermal crescent shift into the interior of the gastrula, they also change their position relative to each other. In the blastula, these two areas lie on opposite sides of the embryo.
Now the lateral horns of the mesodermal crescent converge toward the dorsal side of the embryo and come to lie on both sides of the presumptive notochord. In the next stage that follows the contraction of the rim of the blastopore, the embryo becomes elongated in the anteroposterior direction, all the various presumptive areas participating in this elongation.
The elongation of the notochordal and the mesodermal material brings them into still closer contact with each other, the notochordal material shifting backward, in between the two horns of the mesodermal crescent material.
As a result of these movements, the notochordal material becomes stretched into a longitudinal band of cells lying medially in the dorsal inner wall of the gastrula and flanked on both sides by bands of mesodermal cells similarly stretched in a longitudinal direction. The remainder of the lateral, ventral, and anterior parts of the inner wall of the gastrula consists of endodermal cells.
The external wall of the gastrula similarly takes part in the elongation of the embryo. One of the results of this is that the presumptive material of the nervous system becomes stretched into a longitudinal band of cells lying mediodorsally over the notochordal material but being somewhat broader than the latter.