Detail of cleavage stage during development of the embryo

1. CLEAVAGE
• The process of cell division after fertilization is called cleavage.
• This results the conversion of unicellular fertilized egg into
multicellular animal.
• Characteristics of cleavage are;
1. Unicellular=>multicellular blastomeres(mitotic)
2. No growth
3. No change in shape (Blastocoel)
4. no qualitative and quantitative changes (cytoplasm)
5. Constituents part of cytoplasm not displace to any great extent.
6. Nucleus to cytoplasm ratio changes e.g. in mature sea urchin egg the
ratio is 1/550, at the end of cleavage the same ratio is 1/6.
volume of nucleus 1/550 =>1/6
Volume of cytoplasm

2. • Although the chemical changes during
cleavage are limited but following changes
takes place.
• 1. SYNTHESIS OF DNA
• 2. SYNTHESIS OF RNA
• 3. SYNTHESIS OF PROTEINS

3. Chemical changes during cleavage
• SYNTHESIS OF DNA
• The number of nuclei doubled with every new division.
• It occurs at the expense of synthesis of DNA.
• G1 period becomes limited.
• DNA in synthesized at the expense of cytoplasmic RNA in the
presence of an enzyme Ribonucleotide reductase.
• If the embryo is treated with H3 uridine after some time,
radioactivity will appear in DNA, it means that RNA is involved in
the synthesis of DNA.
• Second source of DNA are the
low density precursors.
e.g: Cytidine is the precursor
for purines.

4. • SYNTHESIS OF RNA
• Synthesis of RNA takes place but it is very
limited, especially synthesis of mRNA takes
place.
• If the egg is treated with Actinomycin D, it will
stop the synthesis of RNA, but the cleavage
will not stop.

5. • SYNTHESIS OF PROTEINS
• Much of the proteins, produced during cleavage are that
which is directly involved in the process of cell
multiplication. e.g. nuclear histones.
• Other protein which is responsible for the formation of
spindle fibre is also formed which is tubulin.
• The third important protein formed during cleavage is
Ribonucleotide Reductase.
• To prove that protein synthesis is important or not treat
the embryo with puromycine which is responsible to stop
the protein synthesis, the cleavage will also stop.
• This proves that protein synthesis is very important.

6. PATTERNS OF CLEAVAGE
• The division of egg into the daughter blastomeres is usually very
regular.
• Generally the plane of first division is vertical i.e. It passes through
the animal vegetal axis of the egg.
• The plane of the second division is also vertical, but it is at right
angles to the first plane of cleavage.
• As a result the first 4 blastomeres all lie side by side.
• The plane of the third division is at right angles of the first two
planes i.e. it is horizontal to the equator of the egg.
• Now there are eight cells.
• Four cells upward and for cells downwards.
• The upper four cells comprising the animal hemisphere of embryo,
and the lower four cells comprising the vegetal hemisphere.
• Pattern of cleavage is different in different animals.
• Some of the main types of cleavage are

7. • RADIAL CLEAVAGE
• If each of the blastomeres of the upper tier lie
exactly over the corresponding lower
blastomeres, the pattern of the blastomeres is
radially symmetrical.
• Radially type of cleavage is found in sea
cucumber (Synapta digitata).

8. Radial cleavage with almost
equal size of blastomeres in sea cucumber
Synapta digitata.

9. • SPIRAL TYPE CLEAVAGE
• In the annelids, molluscs and some of the
planarians the blastomeres are not exactly on
one another.
• The upper cells move spirally.
• If the movement is in clockwise direction, the
cleavage is called dextral type of spiral cleavage.
• If the upper tiers move in anti-clockwise direction
the cleavage is called sinistral type of spiral
cleavage.

10. Spiral cleavage In the mollusc Trochus.

11. • BILATERAL TYPE
• Bilateral type of cleavage is found tunicates and nematodes.
• In these animals two of first four blastomeres become larger
than the other two, thus forming a plane of bilateral
symmetry giving a shape of T.

12. Holoblastic cleavage mammals
- rotational cleavage
Figure 5.9
- first cleavage is meridional
- second cleavage is meridional in one blastomere, but equatorial in the other one
= rotation of cleavage plane
- divisions are NOT synchronized

13. • MEROBLASTIC OR INCOMPLETE CLEAVAGE
• In the eggs of birds & reptiles the cleavage is
always incomplete.
• Only the active cytoplasm divides.

14. Meroblastic cleavage birds
- telolecithal egg
- discoidal cleavage
chicken
embryo
- cleavage furrows appear at animal
pole of the oocyte to form the
blastodisc
- inner cells are continuous with the yolk
- egg is laid during blastoderm stage
Figure 5.15

15. • HOLOBLASTIC OR COMPLETE CLEAVAGE
• In amphibian holoblastic type of cleavage is found.
• whole of the egg becomes subdivided into blastomeres
• SUPERFICIAL CLEAVAGE
• In case of arthropods the division of nucleus takes
place, but the cytoplasm remains undivided.
• In this way a number of nuclei are formed.
• The nuclei later on separate from each other and
arrange themselves at the periphery along with a little
cytoplasm. As a result blastoderm is formed.

16. Meroblastic cleavage insects
Drosophila - centrolecithal eggs
- superficial cleavage
- repeated mitosis without cytokinesis
- multiple nuclei in endoplasm
nuclei move towards the yolkfree periplasm
- pole cells form at posterior pole
- become primordial germ cells
nuclei & cytoplasm form syncytial blastoderm
- cellularization - formation of cellular blastoderm
Figure 5.17

17. Superficial cleavage insects
Drosophila
vitellophages
Figure 5.17

18. Patterns of embryonic cleavage:
holoblastic and meroblastic cleavage
(evenly distributed yolk)
8-7
8-25, 26
8-36, 38
Mesolecithal - amphibians

20. Holoblastic (complete cleavage)
Isolecithal
Mesolecithal
C
Meroblastic (incomplete cleavage)
Radial (echinoderms, amphioxis)
Spiral (annelids, molluscs, flatworms)
Bilateral (tunicates)
Rotational (mammals, nematodes)
Radial (amphibians)
Bilateral (cephalopod molluscs)
Telolecithal
Discoidal (fish, reptiles, birds)
Centrolethical Superficial (most insects)

21. CHARACTERISTICS OF NUCLEI DURING
CLEAVAGE
• Weisman gave the theory of germplasm according to
which there are present certain particles in an egg which
are responsible to determine the cleavage.
• These particles are called determinants.
• He said that these determinants are present on
chromosomes and these may be hereditary material.
• During cleavage these determinants are disturbed in the
blastomeres.
• All these determinant are necessary to develop a normal
embryo.
• It means that all the blastomeres are necessary to
develop into a normal embryo.

22. • Horstadius & Wolsky took the sea urchin egg at 4
blastomeres stage and separate the 4 blastomeres.
• In this way he divides one of the four blastomeres, may
develop into a whole embryo but reduce in size.
• According to germplasm theory the complete set of
determinants is necessary to develop into normal
embryo.
• But they observed that all the four blastomeres are able
to develop into complete individual.
• This is against germplasm theory.

23. • SPEMANN
• He took the fertilized egg of Triturus (newt) and divide it
into two halves with a fine hair, just as they were about to
begin cleave.
• The constriction was not carried out completely in such a
way that the two halves were still connected to each other
by a narrow bridge of cytoplasm.
• Cleavage started in the one half containing nucleus, but the
one half without nucleus remained uncleave.
• At 16 –cells stage he allowed nucleus to go into the other
half which had no nucleus as a result this half also begin to
cleave.
• In a number of cases as a result of this experiment two
complete normal embryos were developed.
• This experiment proves that at 16-cells stage & even 32-
cells stage every nucleus has a complete set of hereditary
factors which are responsible for the normal development.

24. • 2) EXPERIMENT ON DRAGON FLY
• In dragon fly cleavage is incomplete.
• The nucleus divides first, the cytoplasm remains
uncleaved.
• After first division of the nucleus one moves toward the
anterior end where as the other move towards the
posterior end.
• After first cleavage if any of two nuclei is destroyed with
a narrow beam of ultraviolet rays, we find that the other
nucleus continue to divide to form a complete embryo.
• This thing is once again against the germplasm theory.
• This thing can also be further proved by using the
technique of transplantation.

25. • 3). TECHNIQUE OF TRANSPLANTATION
• Take a ripe egg from oviduct of a frog.
• Prick it with a fine glass needle to activate it, and then exclude
nucleus.
• Now take a nucleus from the cell of an advanced embryo with the
help of a pipette.
• Now inject this nucleus in enucleated egg.
• 80% of such egg starts cleaving.
• This experiment proves that nuclei of the later stages have all the
factors responsible for development.
• In further experiments the potential of the nuclei of cells which
were in more advance stage were tested by transplanting then into
enucleated eggs. e.g. Nuclei from the neural plate of well developed
tadpoles of frog were transplanted into enucleated egg.
• Normal embryos were developed.
• In further experiments the potentialities of nuclei of cells, which
were even more advanced in the process of differentiation were
tested.

26. • 4). NUCLEI FROM ADULT TISSUE
• When nuclei from the cells of adult advance tissue
were taken and transplanted into enucleated eggs they
did not show further development.
• If adult cells cultured in vitro, they lose the properties
of different cells and start reproducing mitotically and
if their nuclei are then transplanted into enucleated
eggs, the embryo develop abnormally.
• If nuclei of cells of healthy parts of the abnormal
embryos are transplanted into eggs, the development
of second generation embryos takes place and many
normally developing embryos are produced.

27. • Gurdon in 1968 took the nerve cell (which is highly
differentiated cell and is unable to undergo mitosis)
and transplanted this nucleus in the enucleated eggs.
• He observed that the highly specialized nucleus is
capable to develop into a complete embryo.
• He obtained same results from skin, kidney and
intestine nuclei.
• When the nucleus enters the enucleated egg the size
of nucleus is increased to 30 folds in initial few
minutes and during this interval the nucleus is
changed into the undifferentiated nucleus, and
actually this is the cytoplasm of egg which is capable
to change the fate of nucleus.

28. DISTRIBUTION OF CYTOPLASMIC SUSTANCES IN THE
EGG DURING CLEAVAGE
• For the normal development of the embryo, in the
cytoplasm there are determinants which are important.
• First approach was from Spemann in 1928.
• He was awarded noble prize in the developmental biology.
• Spemann observed that the distribution of pigments is not
equal in the amphibians egg.
• The animal pole is heavily pigmented where as the vegetal
pole is poor in pigment or complete white.
• On one side of the egg the marginal zone broader then
other, and this region is known as the gray crescent.
• This area forms the dorsal lip of blastopore he noted that
after first nuclear division, if we divide the egg into two
parts each part having half grey crescent area and one
nucleus.
• Both parts will develop into normal embryos.

29. • On the other hand if we divide the egg into
two parts in such a way that one half has grey
crescent area and other is devoid of it, the egg
having grey crescent area will develop into
embryo and other will not develop into it.
• It means that the grey crescent has some
importance for the normal development of
the embryo.

30. • The importance of cytoplasmic substances in the
egg for normal development is further proved by a
number of experiments carried on the eggs of
various animals.
• Wilson (1904): The cytoplasm of egg of a mollusc
Dentallium has three zones.
• A clear cytoplasm at the animal pole of the egg
• A second clear cytoplasm at the vegetal pole end
• In between there is a pigmented granular
cytoplasm

31. • After first cleavage the egg divides into two cells. The
division is such that one cell has only two parts i.e.
• Animal clear cytoplasm +pigmented area
• Other cell has all three parts i-e animal clear cytoplasm
+pigmented area+ vegetal clear cytoplasm.
• The reason is that during cleavage the vegetal clear
cytoplasm is pushed off as a polar lobe and remains attach
to the cell CD.
• Cell AB is without this polar lobe.
• If we separate these two cells, the cell containing all three
parts will be able to develop into normal embryo.
• It means all parts of cytoplasm are necessary for normal
development.

32. • At four cell stage only one cell has polar lobe i-e
when AB divides into A and B there is no polar
lobe.
• When CD divides into C and D, the D contains
polar lobe .
• if we separate these four cells only the cell D
having polar lobe will develop into normal
embryo, although nucleus is present in all four
cells.

33. • This is very clear proof that it is the cytoplasm and not
the nucleus that makes blastomere D, different from
other three blastomeres and able to produce normal
embryo.
• This is proved further if we separate the polar lobe of
blastomer ‘D’ then it will also give abnormal embryo.
• Conklin (1905): In the egg of ascidian Styela partita the
eggs has four different zones.
• The animal pole consists of clear transparent zone.
• The vegetal pole consists of slaty grey cytoplasm.
• At the center there are other two zones one consists of
light grey and other is yellow zone.

34. • During cleavage the four kinds of cytoplasm are distributed
to different cells which for different tissues of the embryo.
• Clear zone forms ectoderm
• Yellow zone forms mesoderm
• Slaty grey forms endoderm
• Light grey forms nerve cord +notochord
• He took the egg of Styela and centrifuges it in such a way
that the position of various zones is changed.
• Normally the animal pole is responsible for the formation
of ectoderm.
• But when as a result of centrifuge the animal pole is
dislocated to the lower part (vegetal pole) here it starts to
form ectoderm.
• This clearly determined that fate of embryo is determined
by cytoplasm.

35. BIOCHEMICAL SIGNIFICANCE OF
THESE AREAS
• Muscles are formed from mesoderm.
• The blastomeres which form muscles contain marker enzyme
acetylcholine esterase.
• Dopa oxidase is marker enzyme of the eye and statocysts.
• Both these enzymes appear in the embryo nine hours after
fertilization.
• Treat the dividing embryo with cytochalasin B or with colchicine.
• This will stop cell division but we will see that enzymes will appear
in the correct blastomeres at the fixed time as in normal embryo.
• Now treat the embryo with puromycin, this will stop protein
synthesis.
• Now there is no formation of these enzymes.
• This clearly proves that enzymes must be synthesized during the
development and is not already present in the cytoplasm.

36. • If we treat the embryo with Actinomycin D this
will stop transcription i-e the formation of
mRNA stopped.
• The embryo does not develop the enzymes.
• This proves that transcription process occurring
after 7th hour is very necessary for the
formation of mRNA, which after 8 hours is used
for the formation of enzymes.
• We can further prove that the cytoplasm plays
its role in the egg in determining the
differentiation of cells during development by
the following experiments.

37. • Germ Cells: In insects at vegetal pole there are certain cells called
germ plasms or polar plasm.
• They are called germ plasms because at later stages of
development these cells enter into gonads and form germ cells for
reproduction.
• That is why they are also called “primordial germ cells”.
• To prove this the location of cells in cytoplasm is important?
• Mahowald (1974) transplant these cells to the animal pole.
• He saw that they were unable to enter into gonads although they
are differentiated into polar plasms.
• In the second experiment he transplanted the germ plasms from
animal pole to vegetal pole in another embryo.
• The cells moved & enter to the gonads, and formed germ cells.
• The flies developing from second experiment were mated and they
produced off spring.
• This shows that the location of cells in the cytoplasm is important

38. Meroblastic cleavage insects
Drosophila - centrolecithal eggs
- superficial cleavage
- repeated mitosis without cytokinesis
- multiple nuclei in endoplasm
nuclei move towards the yolkfree periplasm
- pole cells form at posterior pole
- become primordial germ cells
nuclei & cytoplasm form syncytial blastoderm
- cellularization - formation of cellular blastoderm
Figure 5.17

39. ROLE OF THE EGG CORTEX
• In sea urchin, Arbacia the egg contain red pigment.
• We can disturb or change the position of pigment
by the process of centrifugation.
• Morgon (1927) took the egg of Arbacia and
centrifuged it.
• Now the position of the pigment was changed.
• He saw that the process of invagination started at
normal position i-e at the vegetal pole it has no
influence on the site of invagination so the
distribution of pigment is not important for normal
development.

40. • Morgan done another experiment.
• Egg of mollusc Dentallium has three zones
• Two clear zones at upper and lower side and a pigmented zone in
the middle.
• At the vegetal pole, formation of vegetal lobe takes place.
• At vegetal pole yolk is maximum.
• He changed the position of yolk with the help of centrifugation in
such a way that the amount of yolk is now maximum at the animal
pole.
• But the formation of vegetal lobe takes place at the normal position
i-e at the vegetal pole.
• Although the gradients of polar lobe will be different from normal
vegetal lobe (polar lobe).
• So it is concluded that polar lobe is not dependent on the yolk.
• It is clear from the above experiment that the polar factor
responsible for the organization of the cytoplasm is not linked with
• 1) position of the yolk.
• 2) distribution of the pigment

41. • It is also clear that the factor is such which is not
disturbed with centrifugation.
• It is considered that there may be some factors which are
not disturbed with centrifugation
• It may be “skeletal system” but a system of “skeletal
fibre” is not formed in many kinds of eggs.
• The alternative is the cortex of the eggs which is not
displaced by centrifugation
• Many embryologist suggest that the cortex is actually
responsible for the organization of cytoplasm

42. • If we centrifuge the egg of ascidian the whole
cytoplasm is disturbed but the cortex is not
disturbed.
• But later on we see that all the cytoplasm is
reorganized i-e cortex has capability
reorganize the cytoplasm.

43. • In the egg of mollusc Aplysia there are granules which
contains ascorbic acid (vitamin c) probably associated with
Golgi bodies.
• In immature oocytes the vitamin c granules are evenly
disturbed in the cytoplasm.
• During maturation the granules are located on the equator
just under the cortex forming a ring.
• We can easily disturb the layer of vitamin C by
centrifugation.
• After few hours this layer will be rearranged to its original
position.
• We know that normal arrangement of cytoplasm is
necessary for development of normal embryo and the
cortex has capability to rearrange the cytoplasm.

44. Redistribution of vitamin C granules in eggs of Aplysia limacina during maturation
and following centrifugation. a, Immature egg; b, mature egg; c, egg immediately
after centrifugation. d-h, Consecutive stages of recovery of the centrifuged egg,
with the vitamin c granules returning to their normal position.

45. • If we disturb the cortex then normal
development does not occur.
• So the most important part of the cytoplasm
is cortex that has capability to rearrange the
egg of cytoplasmic inclusions and is very
necessary for the normal development of the
embryo.

47. THE MORPHOGENETIC GRADIENTS IN
THE EGG CYTOPLASM
• The cytoplasm and cortex, starts the chain of
reactions which eventually leads to the
differentiation of parts of the embryo.
• The structure of the future embryo is not rigidly
determined by local differences in the cytoplasm
of the egg.
• The local peculiarities of the egg cytoplasm are
only some of the factors necessary for the
formation of organ rudiments.
• e.g. examining the development of the sea
urchin.

48. • At 16-cell stage of the sea urchin, the blastomeres are of
three different sizes.
1. The animal hemisphere consists of 8 blastomeres of
medium size, the mesomeres, form ectoderm.
2. The vegetal hemisphere consists of 4 very large
blastomeres, the macromeres, form endoderm & some
ectoderm
3. and of 4 very small blastomeres, the micromeres, which
lie at the vegetal pole of the egg.
• In a subsequent cleavage the future ectoderm and
endoderm are segregated from each other into an
upper tier of macromeres and a lower tier of
macromeres.

49. • The upper tier, lying immediately under the equator of
the embryo, contains the ectodermal material;
• the lower tier, lying nearer to the vegetal pole, contains
the endodermal material.
• The micromeres develop into mesenchyme, which later
produces the larval skeleton consisting of calcareous
spicules.
• In the sea urchin Paracentrotus lividus, the cytoplasm of
the macromeres possesses red pigment granules.
• The red pigment is already present in the egg at the
beginning of cleavage as a broad subequatorial zone.

51. • BLASTULA STAGE
• In the blastula stage, the cytoplasmic substances are
found in the same arrangement as at the beginning of
cleavage.
• Subsequently, the descendants of the micromeres
migrate into the blastocoele where they develop the
skeletal spicules;
• the descendants of the lower tier of macromeres
invaginate, forming a pocket-like cavity-the gut;
• and the ectoderm produces a ciliary band, serving for
locomotion, and a tuft of rigid cilia at the former animal
pole.
• The ectoderm also sinks inward to produce a
stomodeum, coming into communication with the
endodermal gut at its anterior end, while the original
opening of the pocket-like invagination (the blastopore)
becomes the anal opening.

52. • The embryo of the sea urchin develops into a
larva called a pluteus.
• If the blastomeres of the sea urchin are
separated in the two-cell stage or in the four-
cell stage, each of them develops into a
complete pluteus of diminished size.
• This may be related to the fact that the first two
cleavage planes are meridional, passing through
the main axis of the egg.

53. • All of the first four blastomeres therefore get
equal portions of the three cytoplasmic regions
of the egg (ectodermal, endodermal, and
mesenchymal).
• A different result is observed if the egg is
separated into the animal and vegetal halves
after the third cleavage, so third cleavage is
equatorial or horizontal.
• So the both halves do not have equal properties
of all three kinds of cytoplasmic substances i-e
ectoderm, endoderm and mesoderm.

54. • MORULA STAGE
• If we divide the morula into two halves transversely, the
vegetal pole develops into large endodermal gut and a
small ectodermal vesicle, because it contained the
material of endoderm and a small material of ectoderm.
• The gut is not present inside the ectodermal vesicles
because it is evaginated to the exterior(turned inside
out). This phenomenon is known as exogastrulation.
• The mesenchymal cells produce no skeletal spicules.
• Once again this is due to the fact that both halves do not
have same parts .
• The parts are not going to develop some things because
they do not have material for them.

57. • If the fertilized sea urchin egg (morula) is
treated with lithium (Li) salt in solution, the
embryo once again develops abnormally as in
the previous case.
• There is exogastrulation.
• This is due to the fact that lithium salt
combines with animal pole and makes it
weakened.
• Similarly sodium thiocynate weakened the
vegetal pole and as a result there is no normal
embryo.

58. • From above discussion we concluded that in the
egg of sea urchin there are present two factors
which are antagonistic and yet interact with each
other at the same time and also that
development of normal embryo is dependent on
a equilibrium between these two factors.
• The activities of these factors are maximum at
the poles and decreases as we move away from
the poles, i.e. each has its center of activity at
one of the poles of the egg.

59. • One factor is present at animal pole where its
ability is maximum and decreases onwards vegetal
pole.
• The other factor is present at vegetal pole where its
ability is maximum and decreases towards the
animal pole.
• As far as such kinds of activities are concerned, the
factors are called gradients.
• So there are two gradients animal gradient with
maximum activity at animal pole and vegetal
gradient with a center of activity at vegetal pole.

60. • Normal development occurs when these both are
present in an equilibrium condition.
• If the animal gradient is weakened (lithium salt e.g.)
the vegetal gradient becomes predominant and the
embryo is vegetalized, i.e the vegetal pole develops in
excess such as gut.
• If the vegetal gradient is weakened the animal pole is
predominant and the embryo is animalized, i.e It
develops in excess e.g. ectoderm.
• Ciliary tuft and other structures such as ciliary band
and the skeleton can develop if both gradients are
active and in equilibrium.

61. PHYSIOCHEMICAL NATURE OF ANIMAL-VEGETAL GRADIENTS SYSTEM IN SEA
URCHIN EGGS
• The existence of animal-vegetal gradients can be proved
by using different dyes.
• Slightly stain the embryos of sea urchin with Janus green
(diethyl safranin azo dimethyl aniline) and place them in
a small chamber and sealed them with petroleum jelly.
• Embryos will consume all free oxygen present in the
chamber in respiration.
• Then the embryos will respire using Janus green as an
acceptor of hydrogen.
• The dye will first reduce to a red dye and further to a
colorless substances.
• The light grayish blue (colour of Janus green) first
changes to red i-e (diethyl safranin) and then to a
colorless substance (leucosafranin).

62. • The reduction of the dye takes place in a specific pattern in
the late blastula and early gastrula, the change in colour first
appears at the vegetal pole especially at micromeres which
form mesenchyme.
• In the long run color changes at whole vegetal pole and
reaches the equator.
• At this stage a spot of changed color appears at animal pole
and also gradually increases.
• The red color then begins to disappear in the same sequence,
starting from vegetal pole and then from animal pole.
• If we treat the embryos with lithium salts, this will suppress
the animal pole.
• As a result there will be no reduction in the anima pole.
• Similarly if we treat the vegetal pole by thiocynate sodium, it
will weakened the vegetal pole and there will be no
reduction in the vegetal pole.
• This proves that there are present animal-vegetal gradients in
the sea urchin egg.

63. • Micromeres are center of vegetal gradient
• Micromeres are carrier of the highest point of the vegetal
gradient and they retain this property after
transplantation.
• Accordingly the micromeres can serve as the center of a
reduction gradient.
• If a group of four micromeres is implanted laterally and
the embryo is tested after reduction of Janus green.
• It can be seen in addition to two normal gradients, a
third gradient appears having the implanted micromere
as its center, but spreading out from these to the
adjacent cells.
• It means that micromeres are able to establish a vegetal
gradient in the adjoining parts of blastoderm, we should
expect that they do so by producing some substances
which diffuses into adjacent cells.

64. • Micromere produce RNA while other
blastomeres produce DNA
• After the 4th division of the egg the cleavage is
slow down in the micromeres.
• During this time the macromere and
mesomeres continue rapid division and
synthesize DNA.
• The micromeres in the next division start
synthesizing RNA.
• This can be proved if we use radioactively
tagged uridine, we see only the micromere take
up the labeled uridine.

65. • If the micromere are then transplanted into an
isolated animal half of 16 or 32-cell embryo,
radioactivity can be detected as being widely
spread in the cytoplasm of the host half embryo.
• This clearly shows that the RNA synthesize by
micromeres has diffuse into the cytoplasm of the
adjoining cells.
• This RNA is necessary for gut formation and it
can be proved by the following experiment.

66. • If a 16-cell embryo, instead of being provided
uridine is supplied with 8-azaguanine, an
analogue of uridine, this substance is
incorporated into the RNA by the micromeres. An
abnormal RNA is produced.
• After the treatment the progeny of micromeres,
(the primary mesenchyme) develops normally
but the gut completely fails to develop.
• This proves that the RNA diffusing from the
micromere is essential for gut development and
that if the RNA is abnormal, it cannot perform its
function.

67. • It is suggested that the spreading of substances
from the micromere into the other parts of the
blastoderm occurs directly from cell to cell and
not through the fluid surrounding the cells.
• There are present tiny channels called gap
junctions between the cells.
• Each gap junction is about 20A˚.
• Molecules of mol. wt. over 1000 are passed
through these channels from cell to cell.

68. • The animal and the vegetal gradient in the sea urchin
embryo can be considered to be system of metabolic
reactions which involve oxidations and that these
reactions are maximum at animal and vegetal pole.
• We can also suppress the animal pole (vegetalization) by
using sodium azide and dinitrophenol.
• Sodium azide inactivates the cytochrome oxidase system
and dinitrophenol disturbs the oxidation by preventing
the oxidative phosphorylation i-e there is no formation of
ATP.
• This means the vegetalization is based on disturbance of
oxidative reactions in the embryo especially a
disturbance of phosphorylation.

69. • Lithium salt treatment decreases the oxygen
consumption which occurs normally at the
beginning of gastrulation.
• Furthermore inorganic phosphate accumulates
the sea water during the development of
lithium ion treated embryos.
• This again shows that the embryos are
incapable of utilizing the energy of oxidation for
phosphorylation and synthesis of ATP.

70. • In addition to sodium thiocynate we can
animalize the embryo with the following agents.
• Some metals e.g. zinc, mercury.
• Some proteolytic enzymes: Trypsin,
chymotrypsin.
• Many acidic dyes e.g. congo red, rose bengal,
uranin
• Some sulphonated compounds: germanin
• Some anionic (acid) detergents.

71. • Gradients are protein in nature
• The common property of all these agents is that
they form compounds with proteins especially
basic proteins.
• Their acidic group becomes attach to the
functional group of protein molecules.
• Zinc and mercury also combines to the side
groups of proteins.
• They block the functional site of proteins i.e. they
immobilize the protein molecules.
• In this way the protein molecules cannot interact
with other cells constituents.
• This clearly shows that the gradients are protein
in nature.

72. • In case of animalizing agents the dyes first
stain the primary mesenchyme cells at the
vegetal pole.
• Later on the adjoining presumptive
endodermal cells show the staining too.
• It is thus quite clear that the animalizing is due
to damage to the center of the vegetal
gradient.

73. • A more direct approach involve to isolate animalizing and
vegetalizing substances from the embryo itself.
• By homogenizing sea urchin unfertilized egg or early
cleavage stages and by separation of the materials by
centrifugation and chromatography, some factors were
obtained which can animalize the developing embryos.
• Some vegetalizing activity was also recorded.
• The exact chemical nature of these substance s is yet not
known but the absorption curve of one function in
ultraviolet lights resembles that of amino acid tryptophan
while the absorption of another suggest nucleotide
structure.
• These tryptophan and nucleotide structures are considered
to be animalizing agents.