Name: ___________________________________ Date: _______________

EXERCISE 7
EMBRYOLOGY

Embryology is study of the development of a new individual from an egg. Sexually-reproducing

animals start developing in the formation of a fertilized egg or zygote. This is formed by the
union of the egg cell and the sperm cell. The zygote undergoes cleavage, i.e. a series of mitotic
cell divisions.

When the frog’s eggs are shed into the water during amplexus, i.e. sexual embrace, the male
frog sheds spermatozoa over the eggs. The gelatinous substance deposited on the external
surface of the vitelline membrane, as the eggs pass through the oviduct, absorbs water and
swells and becomes a jelly mass. This increases the size of the eggs.

Fertilization in frogs and toads is external. The effective frog sperm cell always enters the egg in
the animal hemisphere. The sperm contact and penetration of the egg seem to cause the
superficial jelly to swell to its maximum. Another effect of the penetration of the sperm is the
elevation of the vitelline membrane away from the egg and this becomes the fertilization
membrane. This membrane prevents the entrance of another sperm cell. A space appears
between the egg surface and the fertilization membrane which is termed as the perivitelline
space. This is filled with a fluid with which the egg is free to rotate. After the penetration of the
sperm cell there will be the union of the sperm nucleus with the egg nucleus. The egg becomes
fertilized and termed zygote.

The Stages of Embryonic Development:

A. The Fertilized Egg. Eggs of frogs can be gathered from the surface of a canal or pool
during the breeding season, particularly in June or July. Study under a hand lens or
stereomicroscope an uncleaved egg of the frog, i.e. the one-cell stage. It is enveloped in a
thick jelly mass for protection. If the egg is fertilized, a thin fertilization membrane inside the
jelly mass can be recognized. The black portion of the egg is the animal hemisphere while
the colorless portion is the vegetative or vegetal hemisphere. When floating in water, the
heavier vegetal hemisphere is facing the bottom.

Label the parts of the unfertilized eggs below.

 Proceed to https://www.vcbio.science.ru.nl/en/virtuallessons/embryology/xenopus-intoto/ and
examine the fertilized egg. Take a screenshot and label the parts. Draw and label the parts.

The frog egg is classified as _____________ due to the presence of a moderate amount of
yolk which is unevenly distributed.

 continue to proceed to the cleavage stage.

B. Cleavage. Shortly after fertilization, the zygote divides successively by mitosis, so that the
number of cells, now known as the blastomeres increase in geometrical ratio into, 2-, 4-, 8,
16-, 32-cell stages, etc. This period of development is called cleavage.

Examine and draw the whole embryo in 2-, 4, and 16-cell stages
https://www.vcbio.science.ru.nl/en/virtuallessons/embryology/xenopus-intoto/.
Label the blastomeres and cleavage furrow.

Examine the embryo cross-sections of the 2-cell cleavage and morula in

http://sciences.usca.edu/biology/zelmer/122/andev/
Draw and label the animal pole, vegetal pole, blastomeres and cleavage furrow of the 2-
cell stage.
Draw and label the animal pole, vegetal pole, micromeres and macromeres of the morula.

Examine the cross-sections of the 8- cell and late cleavage embryos.

https://sites.newpaltz.edu/histology/developmental-biology/frog/

Draw and label the micromeres and macromeres.

Note: Review your lecture notes or refer to books, for the different stages of cleavage of the
frog’s development and be able to determine the positions of the various cleavage planes in
relation to the animal or vegetative hemispheres.
 Cleavage in frog embryo showing the cleavage planes.

Determine the orientation/location of the following cleavage furrows:

A. 1st cleavage furrow:
B. 2nd cleavage furrow:
C. 3rd cleavage furrow:

Due to the moderate amount and uneven distribution of yolk in the frog’s egg, the cleavage
cells have unequal sizes. This type of cleavage is classified as ________________.

 continue to proceed to the blastula stage.

C. Blastula Stage. As a result of cleavage, the zygote becomes a hollow ball of cells, called
blastula. The cavity is called the blastocoele.

Examine the blastula stage sagittal section

https://www.vcbio.science.ru.nl/en/virtuallessons/embryology/amphibian-section/
And http://sciences.usca.edu/biology/zelmer/122/andev/
Note that the blastomeres in the animal hemisphere are smaller and darker than those of
vegetal hemisphere. The former are called micromeres while the latter are called
macromeres.

Draw the blastula stage embryo and label the micromeres, macromeres and blastocoel.
  continue to proceed to the early gastrula stage (frog crescent blastopore).

D. The Gastrula (Yolk Plug) Stages. The frog’s egg is mesolecithal, that is, the moderate
amount of yolk is concentrated on the vegetal hemisphere. The relative abundance of yolk
and its concentration on one side of the egg makes gastrulation a complicated process. In
the transformation of the blastula into a gastrula, three processes are involved, i.e. epiboly,
involution, and invagination.

The rapid posterior growth of the cells in the animal hemisphere over the cells of the vegetal
hemisphere is a process known as epiboly. The inward growth of the cells in the animal
hemisphere from the outer surface towards the inside is known as involution. The slight
inward growth of the cells in the vegetal hemisphere is known as invagination.

1. Early Gastrula Stage (Early Yolk Plug Stage).

a. Whole embryo, Early Gastrula: Mount the specimen under the stereoscope and
locate a crescent-shaped depression along the line demarcating the animal and
vegetal hemispheres. This is the beginning of the blastopore. Here, the animal
hemisphere is growing over the vegetal hemisphere (epiboly) and at the same time
growing inward (invagination) over the lip of the blastopore. The vegetal hemisphere
is beginning to disappear from the surface of the embryo.

b. Sagittal Section, Early Gastrula: Examine the specimen over the microscope
(LPO). The big cavity in the specimen is the blastocoele. Look for the gastrocoele
(archenteron). This is a small cavity located in one side of the specimen and opens
to the outside through the blastopore which is bordered dorsally by a mass of cells,
the dorsal lip.

Examine the sagittal section of the early gastrula and identify the blastopore,
blastocoel, dorsal lip and archenteron or gastrocoele. Draw and label the parts.
http://www.discoverdevelopment.com/PHP/SlidesFrogEarlyDev.php
  continue to proceed to the late gastrula stage

2. Late Gastrula Stage (Late Yolk Plug Stage).

a. Whole embryo, Late Gastrula: This embryo is now somewhat elongated, and the
component cells are now much smaller. The vegetal hemisphere is now inside
except for a small mass, the yolk plug, protruding out at the blastopore on the
smaller end of the embryo. Look for the dorsal lip, lateral lip, and ventral lip.

b. Sagittal Section, Late Gastrula: Study the section of this stage. Unlike early
gastrula, the blastocoele is now almost obliterated. The now bigger cavity is the
gastrocoele or archenteron. It opens to the outside if it were not for the yolk plug
blocking the blastopore. The embryo is now definitely two-layered (didermic). The
outer layer is the ectoderm and the inner layer is the endoderm. The thick layer of
cells dorsal to the gastrocoele is the chordamesoderm (found underneath the
neural ectoderm). This will give rise to the notochord and mesoderm.

Examine the sagittal section of the late gastrula and identify the blastocoel, dorsal
lip, yolk plug, ventral lip, neural ectoderm, chordamesoderm and archenteron or
gastrocoele. Draw and label the parts.
http://www.discoverdevelopment.com/PHP/SlidesFrogEarlyDev.php

 continue to proceed to the neural plate stage

E. Neural or Medullary Stage.

1. Neural or Medullary Plate Stage.

a. Whole Embryo, Neural (Medullary) Plate. After the complete disappearance of the
vegetal hemisphere and the closure of blastopore, the ectoderm of the dorsal side of
the embryo now flattens into a plate, the neural or medullary plate. It is wider
towards the anterior and narrower towards the posterior.
 b. Cross-section, Neural (Medullary) Plate. Identify the neural (medullary) plate.
Distinguish the three germ layers. The outermost layer is the ectoderm. The
innermost layer is the endoderm, bordering the archenteron, the cavity of the
gastrula. The endoderm is much thicker on the central side. The middle layer, the
mesoderm, does not meet, the notochord being between them. The notochord is the
small mass of cells just below the median portion of the neural plate. In some
specimens, the three germ layers are not yet differentiated in the lower portion
below the archenteron.

Examine the cross section of the neural plate stage embryo and identify the
archenteron, neural plate, ectoderm, endoderm, mesoderm and notochord. Draw
and label the parts.
https://www.vcbio.science.ru.nl/en/virtuallessons/embryology/amphibian-section/

 continue to proceed to the neural groove stage

2. Neural or Medullary Groove Stage.

a. Whole Embryo, Neural (Medullary) Groove Stage. At this stage, the plate is thus
transformed into a groove, the neural (medullary) groove. The borders are raised
into ridges called neural (medullary) folds. The anterior portion of the groove is
broader than the posterior portion.

b. Cross-section, Neural (Medullary) Groove Stage. Note that the lateral edges of
the neural plate are elevated. Identify the neural folds. These are the ridges which
bordered a depression, i.e. the neural groove. The notochord and the three germ
layers, the ectoderm, endoderm, and mesoderm are now clearly distinguishable at
this stage.

Examine the cross section of the neural groove stage embryo and identify the
archenteron, neural folds, neural groove, ectoderm, endoderm, lateral plate
mesoderm and notochord. Draw and label the parts.
 https://www.vcbio.science.ru.nl/en/virtuallessons/embryology/amphibian-section/
http://sciences.usca.edu/biology/zelmer/122/andev/
http://www.discoverdevelopment.com/PHP/SlidesFrogEarlyDev.php

 continue to proceed to the neural tube stage

3. Neural or Medullary Tube Stage.

a. Whole Embryo, Neural (Medullary) Tube. When the neural folds meet and fuse
together on the dorsal side, the groove between them is transformed into a tube, the
neural tube. This tube is lined with the epidermal ectoderm. Externally, only a
longitudinal line marks the fusion of the two folds. Locate for the branchial bars, i.e.
the low ridges on the side of the head region of the embryo. These will develop later
into gills. Look for a pair of protrusions form the ventral surface of the head region,
these are the ventral suckers.

b. Cross-section, Neural (Medullary) Tube. Locate the neural tube. It encloses a

cavity, the neurocoele. The neural tube gives rise to the central nervous system and
the neurocoele will give rise to the ventricles of the brain and central canal of the
spinal cord. Below the neural tube is the notochord, which at this stage serves as
the embryonic support of the body. The biggest cavity seen in the cross-section is
the archenteron. Take note of the three germ layers which are now very distinct.
Notice the lateral region of the mesoderm. It is split into the outer somatic layer and
the inner splanchnic layer. Between these two layers, a cavity (true coelom)
develops. The ectoderm when united with the somatic layer of the mesoderm forms
the somatopleure and the endoderm when united with the splanchnic layer of the
mesoderm forms the splanchnopleure. Look for the closely packed cells, dorsal
and lateral to the neural tube. These are the neural crest cells. They arise from the
dorso-lateral surface of the tube. They will give rise to the dorsal spinal ganglia,
autonomic ganglia, chromatophores, and the medulla of the adrenal gland.

Examine the cross section of the neural tube stage embryo and identify the
archenteron, neural tube, neurocoele, ectoderm, endoderm, somatic layer of the
mesoderm, splanchnic layer of the mesoderm, coelom, and notochord. Examine the
mid-magnification images to see the neural crest cells. Draw and label the parts.
http://sciences.usca.edu/biology/zelmer/122/andev/
https://sites.newpaltz.edu/histology/developmental-biology/frog/
F. The Gill Stages.

1. External Gill Stage. Around 24 hours after the eggs of the frog were fertilized, they
become young tadpoles with gills protruding to the outside. These are called external
gills. The nostrils, the eyes, the mouth and the pair of suckers on the ventral side of the
head, and the prominent tail can easily be identified. The ventral suckers are used for
attachment of the larvae (tadpoles) to the substrate. The muscle segments, called the
myotomes, may be visible through the transparent skin, especially in the tail region.

Examine the whole mount of the external gill stage embryo and identify and label
external gills, nostrils, eye, mouth, ventral sucker, tail, and myotomes.
2. Internal Gill Stage. As the tadpole becomes older, the external gills will be covered by
a thin layer of skin, the operculum. Only a small opening, the opercular aperture,
serves as the exit of the water passing over the gills during respiration. Identify the other
structures formerly seen in the young tadpole. Label the image below.
 Check out the link below to learn more about frog development. It contains line
drawings and wholemount images with label.
https://embryology.med.unsw.edu.au/embryology/index.php/Book_-
_The_Frog_Its_Reproduction_and_Development_2

Group Output

Submit a write-up that answers the following:

1. Define fertilization. Give its significance.

2. Differentiate an egg cell from a sperm cell. Describe them in terms of their anatomy and
functions.
3. Define cleavage. Describe the stages of cleavage in terms of the manner and direction
of the division.
4. List down the fate of the three germ layers, i.e. the different organs that will develop
from each germ layer.