Cell division is an important process in living

organisms. It is involved in the multiplication of cells for

growth and development of an organism. Moreover, cell
division plays a key role in repairing injured body parts
of animals. Hence, processes such as regeneration and

2A
wound healing are affected by cell division.
Cell division can be viewed as a cycle, which can
be arbitrarily, categorized into two stages namely
interphase and mitosis. The interphase, wherein the cell
undergoes growth and prepares for cell division, might
comprise as much as 90% of the cycle. All the
necessary materials needed for cell divisions, which
include microtubule proteins among others, are
synthesized at this stage. Furthermore, to ensure that
CELL DIVISION- each daughter cell would have a complete set of
genetic material, actual replication of genetic material of
MITOSIS DNA occurs in this phase. Once the preparatory stages
are completed the cell undergoes the actual cell division
or mitosis. Although, mitosis comprises 10% of the
cycle, cell biologists consider several phases in mitosis
based on the different events occurring in the nucleus
and cytoplasm of the cell.
In this exercise, the different stages of the cell
cycle will be identified using a whitefish embryo at the
blastula stage. Cells making up this embryo are actively
undergoing cell divisions. Thus, such specimen can
readily show the different stages of the cell cycle.

Objectives At the end of the activity, the students

should be able to:

1. Identify the phases of the cell cycle

2. Identify the structures associated with each
phase of the cycle using the whitefish blastula
as specimen
3. Differentiate the different stages of mitosis
Materials
Prepared slide of whitefish blastula
Light microscope

Procedure
1. Obtain a prepared slide of cross section of whitefish blastula.

2. Focus under LPO. Observe that the section of the blastula contain several
cells revealing different phases of cell divisions.

3. Switch to HPO. Locate and identify the different stages of the cell cycle
using the descriptions below. Mitosis is a continuous process so that it is
likely to see intermediate stages.

A. Interphase: Look for a cell with a distinct nucleus that is with an intact
nuclear membrane. The genetic materials inside the nuclear appear as
thin, thread-like structures appropriately called chromatin. Within the
nucleus is 1 to 2 nucleoli; these are dense, darkly stained bodies
formed by several chromosomal materials that code for certain RNAs.
Near the nucleus is the centrosome which contains centrioles. The
centrioles may not readily be seen using the light microscope.

B. M-phase: Phase during which the cell divides. It is divided into four
stages:

a. Prophase: Locate a cell in which the nuclear membrane starts

to disintegrate or disappear. The chromatin fibers start to
appear as coiled, thickened, condensed structure forming
chromosomes that are visible under the light microscope.
Centrioles are seen in the opposite poles. Locate ray-like
microtubule bodies called asters radiating around each
centrioles and mitotic spindles forming between centrioles. The
nucleoli disappear at this stage.

b. Metaphase: The cell at this stage can readily be identified from

the rest of the phases of mitosis in that the chromosomes are
aligned at the equatorial region of the nucleus. At this stage, the
replicated chromosomes, which are much shorter and more
condensed, are readily recognized. The chromosomes are
made up of chromatids joined together at their central region
called centromere. A protein coat called kinetochore is found in
each of the sister chromatid. The spindle fibers are attached at
the centromere of each chromosome.

c. Anaphase: At this stage, look for a cell where the chromatids

are pulled apart toward the opposite poles. This stage is
differentiated into early and late stages depending on the
distance travelled by the chromatids. Once the chromatids
 separate, these are considered as chromosomes reaching the
opposite poles.

d. Telophase: Look for a cell where a cleavage furrow, a

constriction of the plasma membrane at the region of the
equatorial plate starts to appear. This is the start of early
telophase. At this stage the nuclear membrane and the
nucleolus start to reappear. The chromosomes begin to uncoil
and assume a threadlike appearance. The asters and mitotic
spindles disappear. At late telophase, the cleavage furrow
becomes more constricted leading to the formation of 2
daughter cells.

Cytokinesis, which is the division of the cytoplasm usually, occurs along with
telophase. Completely separated daughter cells are formed soon after mitosis.

4. Be sure that the different stages of the cell cycle are observed and
identified. Do not rely so much on the photomicrograph and diagrams provided;
there is still no real substitute to an actual observation of the specimen.

References

Burns, R.D. and K.A. Stiles. 1997. Laboratory Explorations in General Zoology.
MacMillan Publishing Co., Inc. pp. 15-19.

Campbell, N. 1966. Biology 4th Edition. Benjamin Cummings Publishing House. pp.
207-212.

Dolphin, W.D. 1997. Biology Laboratory Manual 4th Edition. WCB McGraw Hill. pp. 79-
90.

Hickman, F.M. and C.P. Hickman. 1984. Laboratory Studies in Integrated Zoology.
Times Mirror/College Publishing. pp. 26-30.
 Development of organisms may be of two types:
phylogeny and ontogeny. Phylogenetic development
refers to the evolutionary history of the species while
ontogenetic development refers to the entire life history
of an organism. Animal embryology deals with the study

2B of specific stage on the ontogenetic development of an

animal, the embryo. The term embryo, as defined by
most authors (Rugh, 1951; Carlson, 2003), refers to the
developing young from a fertilized egg to a form that is
structurally similar to, but does not yet precisely
resemble, the adult.

The process of change occurring in a developing

ANIMAL embryo is a dynamic and continuous one. The major
stages of development are identified as- zygote
DEVELOPMENT (fertilized oocyte), cleavage, blastulation, gastrulation,
neurulation, and organogenesis. All these stages are
observed in diverse groups of multicellular, sexually
reproducing animals. These stages, however, would
vary in details in different animal groups.

Fertilization involves the union of the sperm and egg

nuclei. This process restores the chromosome number
or diploid number of the organism. The embryo as a
result of fertilization is appropriately called a zygote.
Fertilization in frog occurs externally, that is, it occurs
outside the female body, usually in moist, damp places.
Cleavage is the process of transforming the single
celled zygote into a multicellular entity. This process
involves a series of mitotic division, which transforms
the zygote to several cleavage cells or blastomeres.
The number of blastomeres determines whether it is in
the early or late stage of cleavage. The first mitotic
division results into two blastomeres (2-cell stage). The
second mitotic division yields 4- cell stage. The third
mitotic division yields an 8-cell stage. Subsequent cell
divisions lead to a geometric increase in the number of
blastomeres. However, mitotic divisions at the latter
stages of cleavage occur asynchronously. The ball of
cells that is formed as a result of these series of cell
division is called morula.
Blastulation is the process that involves an internal
secretion of fluid by the blastomeres of the morula. This
process results in the formation of a fluid-filled cavity,
the blastocoel or segmentation cavity. The embryo at
this stage is referred to as blastula. In addition to the
formation of the cavity is the emergence of distinctly two
populations of cells. The layer of cells immediately
surrounding the blastocoel forms the endoderm while the outer layer of cells forms the
ectoderm. At this stage, the embryo is characterized as didermic (di= two, dermis=
layer/coat/skin).
Gastrulation is the stage that involves major cell movements and rearrangements
to form the body plan characteristic of a species. These phenomena are collectively
referred to as “morphogenetic movements”.
Among other things accomplished during gastrulation (gaster= stomach) is the
laying out of the primitive gut, also known as gastrocoel or archenteron. This is the
future digestive tract of the embryo. An opening leading to the gastrocoel called
blastopore is formed in the earlier part of the gastrula stage. The blastopore is the future
anal or cloacal opening in deuterostomes (echinoderms and vertebrates). In
protostomes such as nematodes, mollusks and arthropods, the blastopore becomes the
mouth.
Another important event of gastrulation is the establishment of the three germ
layers namely, ectoderm, mesoderm, and endoderm. These germ layers serve as the
foundation for the organ systems of the animal to take shape. The table below provide
examples of organ system and their corresponding germ layer origin.

Organ Systems/Structures Derived from the Specified Germ

Germ Layer
Layer
Ectoderm CNS, sense organs, epidermis
Mesoderm Heart, muscles, urogenital system, vertebral column, dermis of the
skin
Endoderm Lining of the intestinal tract, lungs

Once the germ layers are established, the different organs start to form. This
process is called organogenesis. One of the organ systems that take shape the earliest
is the central nervous system (CNS). The central nervous system of vertebrates
develops from a dorsally situated ectodermal thickening called neural plate. This
structure invaginates to form a neural fold and a depression called neural groove, which
eventually develops into a hollow neural tube. The process involving the development of
the CNS is called neurulation and the embryo at such stage is referred to as neurula. It
should be noted that other organ systems form during the neurulation stage.
As development proceeds, the embryo gets its nourishment from the stored yolk
or food material from the egg’s cytoplasm. The frog’s egg is of mesolecithal type, which
means it contains moderate amount of yolk. The stored food is depleted as
development continues. The developing young emerge as a swimming larva called
tadpole. Further organogenesis and histogenesis proceed during the tadpole stage. The
tadpole gets its nourishment by feeding on plankton and other microscopic organisms.
The transformation of the tadpole to an adult frog occurs within 2.5 to 3 months through
the process of metamorphosis.
Objectives At the end of the activity, the student should be able to:

1. Identify the different stages of embryonic development in frog specifically,

cleavage, blastulation, gastrulation, and neurulation
2. Identify the different structures associated with the different stages of embryonic
development

Materials

Microscope
Prepared slides of frog’s ovary
Prepared slides of frog embryos
a) Early cleavage
b) Late cleavage
c) Gastrula
d) Blastula
e) Neural plate stage
f) Neural tube stage

Methodology

A. Segmented or Unfertilized Egg

1. Obtain a prepared slide of the frog’s ovary. Focus under scanner or LPO.

2. Locate the spherical cells with well-defined nucleus. Note that the
cytoplasm has granular appearance, usually darkly stained. The cytoplasm
of the egg is filled with yolky material, which serves as nourishment for the
developing embryo. The yolky material is not evenly distributed. It is
concentrated in one area of the egg. The area where the yolky material is
concentrated is the vegetal pole. The area opposite the vegetal pole is
called the animal pole. Such type of egg based on yolk distribution is
referred to as telolecithal type.

B. Cleavage

1. Secure a prepared slide of early cleavage. Focus under scanner or LPO.

2. Locate the cleavage cells or blastomeres making up the embryo. The

outlines or boundaries of the blastomeres can be seen with the furrow or
space in between them. Count the number of blastomeres.

3. Obtain a prepared slide of late cleavage. Focus under scanner or LPO.

4. Find typical embryo at the late cleavage. The blastomeres that are smaller
are sometimes called micromeres and are situated at the animal pole. The
 blastomeres that are larger are called macromeres and are situated at the
vegetal pole. Take note that the vegetal pole is the area where the yolky
material is concentrated. The rate of cell division is hampered by the
presence of yolk. Hence, the blastomeres at the vegetal pole are larger
because of slower rate of cell divisions especially during the latter stages
of cleavage.

C. Blastula

1. Obtain a prepared slide of blastula stage. Focus under scanner of LPO.

2. Orient the specimen such that you can identify the micromeres,
macromeres, vegetal and animal pole.

3. Locate the fluid filled cavity found within the embryo. This is the blastocoel
or segmentation cavity.

D. Gastrula

1. Secure a prepared slide labelled gastrula stage. Focus under scanner or

LPO.

2. Locate the prominent cavity, the primitive gut or archenteron. Find the
opening leading to this cavity called the blastopore. At this stage, notice
that the blastopore is filled with yolk plug. The blastopore is bounded
dorsally by the dorsal lip of the blastopore and ventrally by the ventral lip of
the blastopore.

3. Identify the three germ layers. The outer layer of the embryo is the
ectoderm. The inner layer immediately surrounding the archenteron is the
endoderm. The middle layer between the ectoderm and the endoderm is
the mesoderm.

E. Neurula

1. Obtain a prepared slide labelled neural plate. Focus under scanner or LPO.

2. Locate at the dorsal aspect of the specimen an ectodermal thickening

called medullary or neural plate. This is the early stage in the development
of the neural tube.

3. Secure a prepared slide labelled neural fold/neural groove stage. Focus

under scanner of LPO.

4. Find dorsally the neural folds formed by the ectodermal cells. A depression
or neural groove is seen bounded by the neural fold.
 5. Obtain a prepared slide of neural tube stage. Focus under scanner or LPO.

6. At this stage, the neural folds meet at the center and move inward to form
a neural tube. The neural tube serves as the precursor of the brain and
spinal cord. Locate a round structure immediately below the neural tube.
This is the notochord, which serves as the main axial support of vertebrate
embryos.

7. Find a cavity immediately below the notochord. This is the archenteron or

the primitive gut. The lining of the gut is the endoderm.

8. Locate lateral to the notochord, mass of cells, which make up the

mesoderm. The mesoderm is divided into segments or somites. The most
dorsal somite is the epimere. This is situated at the level of the notochord.
The middle portion of the mesoderm forms a string of cells, which is the
mesomere. This is origin of the urogenital system. Note that the mesoderm
extends ventrally towards the guts. The ventral most portion of the
mesoderm is the hypomere or lateral plate mesoderm. Notice that this
portion of the mesoderm is subdivided into inner splanchnic layer and an
outer somatic layer. A cavity is formed between these two layers which is
the future coelom or body cavity of the animal.

Identify the different stages and label the parts found in your Activity Sheet.

References

Hickman, C.P., L.S. Roberts and A. Larson. 2001. McGraw-Hill Book Company.

Rough, R. 1951. The Frog: Its reproduction and development. McGraw-Hill Book,
Company.

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