CLEAVAGE

AND FORMATION OF THE BLASTULA

Cleavage
Ø Waves of cell division follow one another almost
without pause
Ø Subdivide the large zygote into progressively small
cellular units
Ø Cells from the first few cleavage division
a) Most part morphologically unspecialized
b) Remain metabolically unspecialized
c) Synthesis activity is geared toward the
production of DNA and proteins required
for cell division rather than toward
specialized molecular activity
The cell during cleavage
Ø Cytokinesis occurs after karyokinesis
Ø Plane of the cleavage furrow is the same as that of
the metaphase plate of the preceding mitotic figure
Ø According to Rapport:
a) The position of the cleavage furrow is
established by the time of anaphase
b) After anaphase the mitotic apparatus can
be removed or destroyed without altering
the location or course of cleavage furrow
formation
Ø Asters
• Composed of microtubules
• Structures that interact with the cell
cortex to stimulate the formation of
cleavage furrow
Ø At the site of cleavage furrow, the cell cortex
contains a band of microfilaments
Ø Microfilaments
• Belong to the actin family of contractile
proteins
• In conjunction with myosin molecules in
the same area
• Contractile behavior appears to be the
basis for the constriction of the cell during
cleavage
• As the cleavage furrow spreads toward the
vegetal pole, its rate formation decrease as
a result of the retarding effect of the yolk
• Net result is the appearance of later and
larger blastomeres at the VP (vegetal pole)
than the AP (animal pole)
Ø Holoblastic cleavage – complete division of cells
Ø Telolecithal
• Egg with large amount of yolk
• Displaces the embryo-forming cytoplasm
into a small disk on one edge of the ovum
Ø Meroblastic
• Incomplete division of cells
• When cleavage begins, membranous
material appears along the lateral surface
of the newly forming blastomeres, but
initially it does not separate their inner
borders from the underlying yolk
Cleavage and formation of the blastula in Amphioxus
Ø Amphioxus – primitive chordate, undergo a very
regular form of equal holoblastic cleavage in which
there is relatively little difference among the
blastomeres
Ø First cleavage division: cuts the egg in half from
the AP to the VP in the plane of the metaphase
plate of the dividing nucleus
Ø The nuclei of the two blastomeres form mitotic
spindles at right angles of the plane
Ø Second cleavage division: proceeds again from AP
to VP (4-cell embryo)
Ø Third cleavage division (equatorial division): cuts
each of the four blastomeres in half, roughly
midway between the AP and VP
Ø Fourth cleavage division: produces simultaneous
vertical planes of cleave that cause the embryo to
double its cell numbers
Ø Morula stage: 32-cell stage
Ø Blastula – when a cavity forms

Distribution of yolk and its effect on cleavage

Ø Refer to the table below!!!
Ø Blastomeres – cells that arise from cleavage
Ø Oligolecithal – eggs with little yolk
Ø Mesolecithal
• Egg with moderate amounts yolk
• Yolk tend to be concentrated toward the
vegetal pole
Type of egg (base on Type of Pattern of Representative Blastula Blastula cavity
yolk content) cleavage cleavage animal groups
Oligolecithal- Holoblastic Radial Echinoderms, Sphere; wall a single Large central
isolecithal Amphioxus layer sphere
Bilateral Ascidians
Spiral Mollusks, annelids
Rotational Mammals
Mesolecithal Holoblastic Radial Amphibians, lampreys, Sphere; wall layered Small accentric
lungfish and of nonuniform sphere
thickness
Telolecithal Meroblastic Discoidal Most fishes, reptiles, Cell disk on surface of Flat space
birds yolk between epiblast
and hypoblast
Centrolecithal Meroblastic Superficial Insects and other Yolk-filled cylinder None
arthropods
 CLEAVAGE AND FORMATION OF THE BLASTULA
Cleavage and formation of the blastula in Sea
Urchins
Ø Rapid process, initially consisting of only two
phases of the cell cycle
1) DNA synthetic phase (S)
2) Mitosis phase (M)
Ø Early cleavage: periods of cleavage alternate with
periods of cyclin synthesis and degradation
Ø Late cleavage: G2 then G1 phases become evident
Ø Pattern of cleavage is more complex than
Amphioxus
Ø First two divisions: meridional (AP to VP)
Ø Third cleavage division: equatorial (8-cell embryo
with blastomeres of equal sizes)
Ø Fourth cleavage division:
• Results in the production of three distinct
types of blastomeres
• Animal blastomeres undergo meridional
cleavage è mesomeres
• Vegetal blastomeres divide
asymmetrically è macromeres and
micromeres (below)
Ø Formation of micromeres: dependent on the
presence of cytoplasm that was located at the
vegetal pole
Ø If vegetal cytoplasm removed = micromeres do not
form
Ø Cleaving sea urchin embryo is divided into five
territories:
1) Prospective oral ectoderm
2) Prospective aboral ectoderm
3) Prospective skeletogenic mesoderm
4) Vegetal pole
5) Small micromeres
Ø Each territories are derived from specific
segregated set of founder cells and are
characterized by unique patters of gene expression
and individual cell lineages
Ø Embryo is enclosed in the fertilization membrane
and the outer surfaces of the blastomeres are
closely associated with the hyaline layer
Ø Hyaline layer – formed as a result of the emptying
contents of the cortical granules into the
perivitelline space during fertilization
Ø Seventh and eight cleavage division: central cavity
(blastocoel) becomes well established and embryo
has entered the blastula phase
Ø Wall of blastula is only a cell layer thick and all
cells have an apical surface exposed to the outer
hyaline surface and a basal surface exposed to
blastocoel
Ø Early blastula: spherical; difficult to locate the
original AP and VP
Ø Tenth cleavage division: blastomeres form motile
cilia which penetrate the hyaline layer and extend
into the perivitelline space
Ø Cells of the blastula secrete into the perivitelline
space a hatching enzyme which digest the
fertilization membrane
Ø Ciliated blastula is freely swimming in the sea
Ø Long, nonmotile cilia at the AP is then form
Ø At the same time, region around the vegetal pole
flattens to form the micromere-containing vegetal
plate
Ø Final major event during the blastula phase:
former micromeres make their way into the
blastocoel
Ø These cells, which will form the primary
mesenchyme, first become elongated by the
elaboration of parallel bundles of microtubules
Ø As it elongates, their apical surfaces detach from
the hyaline layer
Ø They next pass through the basal lamina and into
the blastocoel where they rest along the inner
surface on the basal lamina and become readily
recognizable as the primary mesenchyme
Ø Embryo is then often called mesenchyme blastula
Cleavage in the Ingression of primary
Sea urchin embryo mesenchyme cells in the
sea urchin embryo
Cleavage and formation of the blastula in
Amphibians
Ø Completed within 24 hours
Ø First cleavage division: begins at the AP and
bisects the gray crescent
Ø In the axolotl, the cleavage furrow elongates at a
rate of about 1mm/minute in the AP but slows to
0.02 to 0.03 mm/min as it nears the VP
Ø Second cleavage division: begins at AP, with its
plane perpendicular to that from the first cleavage
plane
Ø Third cleavage plane is horizontal and passes
nearer to the AP, dividing embryo into four small
blastomeres at the AP and four large blastomeres
at the VP
Ø Successive cleavage divisions follow one another
rapidly and synchronously
Ø Morula – an embryo between the 16 and 64-cell
stage
Ø In subsequent cleavage cycles the waves of cleavage
begin to lose their synchrony because of a
lengthening of the cell cycle in the cells of the VP
Ø Fifteenth cleavage division (in axolotl): cleavage at
VP is delayed about two cycles compared to AP
Ø After Morula stage, blastocoel appears in the AP
above the mass of yolk
Ø Formation of blastocoel:
 CLEAVAGE AND FORMATION OF THE BLASTULA
• A specialization of the cleavage furrow at
the AP leaves a small intercellular cavity,
which is sealed off from the exterior by
close junctions between the two
blastomeres
• Maintenance and accumulation of fluid
seems to be due to the pumping of Na+
from the blastomeres into the emerging
blastocoel
• Wall of blastula (semipermeable
membrane), water enters the blastocoel to
maintain an ionic balance, causing
blastocoel to expand
Ø Blastula – an embryo that completed the cleavage
cycle from the 64-cell stage to the 128-cell stage
Ø Embryo remains in the blastula stage until
successive cleavage cycles have increased the cell
number between 10,000 and 15,000 blastomeres
Ø Three main regions of blastula:
1) A region around the AP: includes cells
forming the roof of the blastocoel (future
ectoderm)
2) A region around VP: includes large cell in
the interior which constitute the yolk mass
(future endoderm)
3) Marginal ring of cells in the subequatorial
region: region of the gray crescent
(embryonic mesoderm)
Ø Nieuwkoop – discovered the basis for mesodermal
induction
• Mesoderm in the amphibian blastula
forms in an equatorial ring between
prospective ectoderm and endoderm
• Nieuwkoop performed recombination
experiments in which a sheet of cells from
the AP above the blastocoel was directly
apposed to the yolk mass from the VP
• Under an inductive influence from the yolk
mass, the cells from the AP formed
mesodermal structures
• He postulated that one of the functions of
the blastocoel may be to restrict the
interaction between future endodermal
and ectodermal cells to the marginal ring
surrounding the edges of the blastocoel
Ø Mesodermal inducing center
• Source of mesodermal induction
• Resides in a small number of vegetal
endodermal cells located in the prospective
dorsal midline as early as the 32-to 64-cell
stage
• Not only stimulates the formation of
mesoderm, but also establishes the dorsal
properties of the induced mesoderm
• Dorsal induced mesoderm is the
forerunner of Spemann organizer (the
dorsal lip of the blastopore)
Ø VgI (Vg = vegetal)
• A posttranslationally processed protein
• The natural mesoderm-initiating
substance, dependent upon activin
• Inducing effects are directed toward
ventral rather than dorsal structures
• Inducing activity with activin stimulates
the expression of several mesoderm-
specific genes
• Ex. Goosecoid and noggin – discovered in
Drosophila; secondarily induced
dorsalization and come into prominence
during gastrulation
Cleavage
In
Frog’s egg
Cleavage and formation of the blastula in Birds
Ø Entire period of cleavage occurs as the egg is
passing down the oviduct
Ø By the time egg is laid, early gastrulation has
already begun
Ø Newly fertilized egg contains a whitish germinal
disk about 3mm in diameter at the AP
Ø First cleavage furrow: begins to appear near the
center of the blastodisk during late anaphase of the
first mitotic division after fertilization
Ø The sequence of avian cleavage is not always
regular and after about the third cleavage division
it is not synchronous
Ø Fourth cleavage furrow: circumferential; cuts a
central row from a peripheral row of blastomeres
Ø First few cleavage division: blastomeres are
unusual in having their tops and sides bounded by
plasma membrane but their basal surfaces open to
the underlying yolk
Ø Blastoderm – further cleavage in the early disk of
embryonic cell; results in the radial extension of the
embryo
Ø 32-cell embryo:
• Cleavage planes shows an entirely
different character
• Cleavages appear below the surface and
parallel to it
• Establish a superficial layer of nucleated
cells which are completely delimited by
plasma membranes
• Superficial cells rest on layers of cells
which on their deep faces are continuous
with the yolk
• Continuous divisions of the same type
establish several strata of superficial cells
Ø When embryo contains about 100 cells, the
blastoderm is underlain by a subgerminal cavity
Ø Subgerminal cavity:
• pH (6.5) lower than the albumen (9.5)
leading to the establishment of a
transepithelial electrical potential of 25
 CLEAVAGE AND FORMATION OF THE BLASTULA
mV between the ventral (+) and dorsal (-)
sides of the blastoderm
• Electrical gradient (transport of Na+ and
H3O from apical to basal side), may
determine polarity of the blastoderm
• Reversing the pH gradient or applying an
electrical potential gradient of opposite
polarity to the blastoderm = reversed
dorsoventral axis
Ø First few days of development:
• Blastoderm expands over the surface of the
yolk (involves an active migratory process)
• Cells at the edges are attached to the
overlying vitelline membrane and use this
as a substrate for their migration
Ø After a number of cleavage cycle, the shedding of
individual cells begins from the undersurface of the
area of the blastoderm that is farthest away from
the source of gravity
Ø Area pellucida – central portion of blastoderm,
thinned out by the shedding of cells and underlain
by the subgerminal cavity
Ø Area opaca – surrounds the area pellucida; region
where the cells of the blastoderm still abut directly
onto the yolk
Ø Time the egg is laid: individual cells or aggregates
of cell shed from the lower surface of the
blastoderm by a process of polyingression coalesce
to form primary hypoblast
Ø Process occurs at the posterior end of the embryo
Ø Primary hypoblast
• Disklike layer
• Separated from epiblast (outer layer of
blastoderm) by blastocoel
• Forms extraembryonic endoderm
• Possesses an inherent polarity
• Polarity and location determine the
location and direction of the future
primitive streak by a form of inductive
interaction
Ø Comparison with amphibian blastula:
• Epiblast = AP of the amphibian blastula
• Epiblast remains competent to react to the
influence of the hypoblast by forming the
mesoderm (mesoblast)
• Primary hypoblast share many common
properties with the VP of the amphibian
embryo
Surface
aspect of
bird’s
egg
Blastula of (A) Amphioxus, (B) Amphibian, and (C, D) Chick
Cleavage and formation of the blastula in Mammals
Ø Early cleavage divisions: unmodified mitoses (equal
holoblastic cleavage of an isolecithal egg)
Ø Studies on cleavage were largely conducted in pig
embryos
Ø In recent years, it has been directed toward
primate and mouse embryos
Ø Primate embryos: for the importance of in vitro
fertilization techniques in humans
Ø Mouse embryos: for its genetics and development of
methods allowing studies of cell lineages and cell
determination
Ø Cleavage in mammal is slower
Ø First cleavage division:
• Not completed for 24 hours
• The plane of first cleavage division
includes the polar bodies
• Point of reference: polar bodies are given
off
Ø Second cleavage division:
• May not occur simultaneously in both
blastomeres
• Results in temporary appearance of three-
cell stage
• In many mammals, mitotic spindle of one
of the blastomeres rotates 90° (results in
crosswise arrangement of blastomeres at
the four-cell stage)
Ø Compaction
• A critical stage that takes place at the
eight-cell stage in the mouse
• Blastomeres flatten and become slightly
joined so that they cannot become
distinguished from one another with the
light microscope
• Intercellular connections (dominant
feature) serve two purposes:
 CLEAVAGE AND FORMATION OF THE BLASTULA

1) Tight junctions – prevent the free Ø “Inside-outside” hypothesis: cells of the morula
exchange of fluid between the which have no contact with the exterior develop in
inside and outside of the embryo, a unique microenvironment created by the external
allowing accumulation of fluid cells
inside the embryo Ø Polarization/cytoplasmic segregation hypothesis:
2) Gap junctions – couple all the relates the differentiation of the two cell types to
blastomeres of the compacted gradient of cytoplasmic determinants which become
embryo and permit the exchange segregated into internal or external blastomeres as
of ion and small molecules from cleavage divisions proceed
one cell to the next Ø Relationship of mammals with birds:
Ø 16-cell stage: enclosed in the zona pellucida, and in • Mammalian embryo form layer of cells
the morula stage beneath the inner cells = primary
Ø Internal secretion of morula by the blastomeres in hypoblast of the avian embryo
the morula results to the formation of blastocoel or • This layer will become the primitive
blastocyst cavity endoderm
Ø Changes in the transition from morula to • Primitive endoderm in mouse contribute to
blastocyst: the formation of yolk sac
1) Rapid enlargement of the blastocyst cavity
2) Emergence of distinctly different types of
cells within the embryo
Ø Fluid in blastocyst:
• Product of a sodium transport system
involving polarized Na+/K+- ATPase that
develops in the outer blastomeres
• Brings Na+ with water into the spaces
among the inner blastomeres in exchange
for intraembryonic H+, which is
transported out from the embryo
• Uteroglobin and other uterine proteins:
found within blastocyst fluid of rabbit
embryos
Ø Early mammalian blastocyst remains enclosed
within the zona pellucida
Ø Populations of cells in blastocyst: Cell junctions during cleavage in
1) Trophoblast Cleavage in pig’s embryo mouse embryo
o Cells that constitute the outer
wall of the blastocyst
o Assumed the configuration and
many properties of epithelial cells
o Cells can both pump fluid and
induce special changes in the
Early development
uterine lining upon implantation
of the mouse
o Maternal X-chromosomal genes
are expressed and paternal genes
are inactivated
o Cells form a large part of the
placenta
2) Inner cell mass
o A small group of cells on the inner
surface of the trophoblast
o Joined to one another by
communicating gap junctions Organization & properties of the Embryo during Cleavage
o Retain the ability to reaggregate and Blastulation (Experimental Embryology)
if separated or mixed with the Ø Hans Driesch
cells of other embryos • Isolated blastomeres from two- and four-
o Can neither pump fluid or evoked cell sea urchin embryo and found that a
the decidual reaction perfect pluteus larva formed from each
o Destined to from the embryo plus blastomere
some of the membrane associated • “Prospective potency of the early
with it blastomeres is greater than their
Ø Position of the blastomeres in the morula prospective fate”
determines whether it will become part of the • The property of a part being able to form a
trophoblast or the inner cell mass whole is a good example of embryonic
regulation
 CLEAVAGE AND FORMATION OF THE BLASTULA

• Harmonious equipotential systems: Ø In mouse, blastomeres of four-cell embryo have
systems possessing the kind of property been shown to be totipotent
mentioned • Individual blastomeres or pair of cells
Ø Not all types of embryos are capable of derived from single blastomeres have been
compensating for defects by regulation combined with blastomeres of genetically
Ø Mosaic development: different hosts
• Different response to defects exhibited in • Totipotency: cells are capable of entering
groups such as mollusks either the trophoblastic or inner cell mass
• An isolated blastomere forms little more Ø Tetraparental (allophenic) mice – produced by
than what it would have formed if left in removing the zona pellucida from cleaving embryos
situ and then combining the two embryos
Ø Slicing either the egg or the embryo into two parts
(ex. sea urchin) through a plane including the AP Molecular events during Cleavage and Blastulation
and VP still give rise to normal larvae Ø Merogones
Ø But when bisected through the equatorial plane, • Enucleated fragments of sea urchin eggs
the progeny of the two halves are different • When parthenogenetically activated,
Ø Vegetal half è undergoes regulation cleavage divisions showing a remarkable
Ø Animal half è exhibit mosaic properties degree of regularity ensued, and structures
Ø Dauerblastula (permanent blastula) – form if the showing the external features of blastulae,
cells of the animal hemisphere are separated from but without blastocoel, developed
the vegetal cells and allowed to develop Ø Actinomycin D
Ø Normal looking pluteus larva – animal hemisphere • In certain doses act as a general inhibitor
plus micromeres of RNA synthesis
• Add to sea urchin: cleavage continued
unabated until the embryo had approached
the early blastula stage
Ø Puromycin
• Add to sea urchin and amphibian: cleavage
Experiments quickly ceased
demonstrating Ø RNA synthesis is not required for early cleavage
the importance divisions to occur
of a vegetal Ø (BUT) Absence of protein synthesis further
influence in the development comes to rapid halt
completeness of Ø RNA synthesis in amphibians:
regulation of
components of
• Very little RNA is produced until just
sea urchin before the blastula stage
embryos • As gastrulation begins. Members of rRNA
family become synthesized in increasing
amounts
Ø RNA synthesis in echinoderms:
• At third or fourth cleavage division:
synthesis of mRNA and heterogeneous
nuclear RNA begins
Ø Spemann • New rRNA and tRNA appear around the
• Conducted experiment to determine time of the blastula
whether the nuclei of blastomeres retain Ø Maternal RNAs
the potential to guide the entire course of • Expressed during cleavage
development of whether their capacities • Become unmasked within 30 minutes after
become restricted fertilization
• Constricted a fertilized amphibian egg • Soon join with stored-up ribosomes to form
with a hair loop so that one lobe of the egg polysomes
contained the nucleus & some cytoplasm • Cyclins
and the other lobe with cytoplasm only o Coded for by maternal RNAs
• 160-cell stage: he loosened the ligature and o Synthesized after each cleavage
allowed nucleus to move over into the lobe division
of nonnucleated cytoplasm o Accumulated at high levels by the
• It then went to develop and the other lobe middle of the cycle
without nucleus lost its capacity to direct o Destroyed with the succeeding
embryonic development round of cleavage division
Ø Identical twins can result from regulation and o At the time of blastulation,
normal development of blastomeres that have prominence decreases
become separated during early cleavage
 CLEAVAGE AND FORMATION OF THE BLASTULA

• Histones – another prominent class of
proteins that is synthesized on maternal
messages during early cleavage
Ø Protein synthesis in mammals:
• First cleavage division: early proteins are
made from maternal mRNAs
• Synthesis of histones is prominent during
cleavage
• Cavitation, compaction and formation of
blastocyst is under the control of the
embryonic genome
Ø Mammalian embryos differ from amphibian
embryos in their energy requirement
• Amphibian embryo: must be self-contained
unit with respect to energy sources
• Mammalian embryo: depend on continuing
supple of energy from their surroundings
in both the uterine tubes and uterine
cavity