25 STUDY OF PREPARED SLIDES OF

THE DEVELOPMENTAL STAGES OF

FROG

25.1 INTRODUCTION

Animal development begins usually with the fertilization of an egg by a sperm. The
nuclei of the egg and spenn fuse, and the male and female parent's genes determine the
characteristics of the offspring. Compared to other biological processes, embryonic
development is relatively slow. New cells, tissues and organs make their appearance in
the embryo over a period of hours, days or weeks.
In this laboratory exercise, we shall study the development of frog with the help of
prepared slides and stained sections, from the unfertilized egg to the tadpole stage.
However, before we do so, in order to make the exercise more meaningful, we will
begin with a brief description of the major events in the development of frog. You may
also read units 13, 14 and 15 of Block, 3 of LSE-06course.
Description of Frog Embryology
The frog is an semiaquatic animal and deposits its eggs in water. The egg is of
mesolecithal type having a moderate quantity of yolk which is concentrated in the
vegetal (lower) hemisphere. Fertilization occurs minutes after ovulation and is essential
for further development to proceed. The developmental stages from ovulation until
tadpole stage occurs in water. The ovum has polarity even before fertilization and is
enclosed in three jelly layers. The egg itself has two distinct regions (1) The animal
hemisphere-the darkly pigmented region, which occupies more that half of the egg and
contains the nucleus and (2) the vegetal hemisphere the light coloured, yolk filled
heavier region (Fig. 25.1).
Fertilized e g e y g o k ) - The entry of the sperm into the egg, results in the formation of
zygote. The sperm can enter only at the animal pole and when it does so, usually a grey
crescent forms on the side opposite the sperm entry (Fig. 25.2). On the entry of sperm
other changes also occur (Refer to unit 13 of LSE-06course). The vitelline membrane,
bound tightly around the egg lifts away, allowing the zygote to rotate so that animal
pole becomes uppermost if it was not so before.
Early cleavage and morula formation - Development is rapid after fertilization so
that within a few hours at normal temperature the zygote cleaves or divides into many
cells (blastomeres). Cleavage is holoblastic (entire egg cleaves) and unequal; that is the
blastomeres are not of equal size. Those at the animal pole being smaller in size are
called micromeres and t h ~ s eat the vegetal pole are called macromeres. Early cleavage
results in a solid ball of blastomeres called morula. The total cluster of blastomers is
still no larger than the original fertilized cell.
Blastula -After about the fourth or fifthcleavage, a cavity called blastocoel develops
from the morula giving rise to a blastula. This fluid filled cavity is more towards the
animal pole and can be only viewed in a section of the embryo. The division of the
blastomeres is no longer synchronous (Fig. 25.3). The blastomeres towards the animal
pole divide more rapidly than the yolk laden ones present at the vegetal pole. Thus
externally the blastomeres of the animal pole cover most of the blastula.
Gastrula - Gasuula appears as a dark pigmented ball of cell~~with a circular yolk plug
of larger vegetal cells. A gasuula is formed when the single layered blastula is
transformed into a three layered suucture with an (i) outer ectoderm. (2) intermediate
mesoderm and (3) an internal endoderm. Gasuulation involves division and movement of
cells and these activities follow a &fmite pattern &pepding on the amount of yolk (Fig.
25.4a).
Formation of gasaula begins by the pushing in or invagination of the cells at the animal
pole at a position somewhat below the equator of the blastula, at the boundary of the
animal and vegetal hemispheres in the area of the grey crescent. The invagination forms
a crescent shaped lip and is called blastopore. The position of the blastopore marks the
Laboratory Course I1 future posterior end of the embryo. The future anterior end of the embryo will develop
at the animal pole. The cells of the animal pole continue to divide and move around the
embryo (epiboly) and then turn inward from the blastopore (invagination or involution).
A new cavity the archenteron is formed which expands at the expense of the blastocoel
and is connected to the blastopore. The archenteron later develops into the gastrocoel.
'
After the completion of all the events mentioned so far, it is possible to identify the
three primary layers of the gastrula in a vertical section of the embryo (Fig. 25.4b).
Neurula -Near the end of gastrulation, there is a thickening of the ectoderm along a
longitudinal axis on the dorsal surface to form a noticeable flattened saip called the
neural plate. The edges of this neural plate fold upward and toward each other, finally
to form a closed tube called the neural tube in the midline (Fig. 25.5). The neural tube
of ectodennal cells running from the anterior to the posterior end of the embryo
detaches and sinks below the ectoderm (which was previously [Link] it). The
neural tube thus gets covered by the epidermis and in time differentiates into brain and
spinal cord.
Meanwhile the notochord also develops from a row of cells (of mesodermal origin) in
the mid-dorsal wall of the archenteron as a cylindrical rod and comes to lie beneath the
neural tube and above the archenteron. During this time, mesodennal tissue ventrolateral
to the notochord and distinct from the notochord also develops laterally. The mesoderm
splits into splanchnic and somatic layers within the body cavity. The coelom lies
between these two layers.
The morphogenetic movements of gastrulation and neurulationbring various groups of
cells into position so that the ground plan is laid for their subsequent interaction and
differentiation into the final tissues of the adult in later developmental stages. The head
begins to form'at the anterior end. The mouth breaks through the anterior end of the
archenteron and gill slits develop in the lateral walls of the archenteron. A tail bud
forms at the posterior end which develops into a tail.
The neurula thus gets transformed into a young larva or tadpole with four distinct
characteristics of all chordates; a tube-within a tube body plan, dorsal nerve cord.
notochord and gill slits (Fig. 25.6).
Free swlmming larva -The larva or young tadpole soon becomes capable of
independent existence and emerges from the jelly mass or gelatinous covering to become
a free swimming organism. At this stage the herbivorous larva no longer depends
entirely upon its store of yolk for nutrients. Furthermore a fold of skin called
'operculum' grows backwards and covers the internal gills.
After growing for a few months, the Iarva metamorphoses into %adult. This process
involves marked external and internal changes (Fig. 25.7, 25.8, 25.9). The mouth, teeth
and tongue change as the herbiviorous larva becomes a carnivorous frog. Circulatory
modifications accompany the change from gill to lung circulation. The musculature
adapts from that for swimming in a fish-like manner to locomotion using the limbs.
Objectives
After completing this laboratory exercise you should be able to:
identify and describe the representative stages in frog development from fertilized
egg upto tadpole stage is observed in microscopic preparations.
draw and label the early and late embryonic as well as the larval stages in the
prepared slides.

MATERIALS REQUIRED
-
prepared microscope slides of frog development.
unfertilized egg
blastula enlire and its vertical section
gastrula entire and its vertical section
neurula entire and its vertical section
whole mount of tapole larva of frog
I .[Link] ear of tadpole Study of Prepapd Slides of the
Developmental Stages of Frog
T.S. through eyes of tadpole
T.S. through the head and gills of tadpole
microscope.

PROCEDURE
Before you start studying the slides, carefully go through the description of frog
embryology given in the introduction and the various figures provided throughout the
laboratory exercise. This will help you to become familiar with the various
developmental processes occurring in frog and to relate them with the embryological
slide material you will be studying. First by examining whole mounts of embryonic
stages become familiar with the general and overall changes taking place in frog
development. Make drawings of these stages in your record notebook by observing them
under the microscope. This study should be followed by the study of the sections of the
developmental stages and you should be able relate them to the study whole mounts.
Your counselor will arrange the slides of various stages of frog development. Examine
the slides carefully first under the low power and if necessary under the high power of
the compound microscope and compare what you have observed with the description
and figures provided in the lab exercise. After viewing one slide you may move to next
until you complete viewing the entire series.
,In you notebook sketch, label and write the description of all the stages of the
development which you have observed.
The stages which you will be examining and for which descriptions are provided are
listed below:
1. Unfertilized egg
2. Fertilized egg
3. Blastula entire and V.S. of blastula
4. Gastrula entire and V.S. of late gastrula
5. Intact neurula and V.S. of neurula
6. Tadpole larva of frog
7. T.S. of tadpole through eye region
Pigmented layer
8. T.S. of tadpole through auditory vesicles (ears)
Vitelline membrane
9. T.S. of tadpole through head and gills.
Future animal poie 1
A Unfertilized Egg of Frog
1. The egg is moderately telolecithal or mesolecithal
2. The egg or ovum is surrounded and covered by three distinct layers (a) the
innermost vitelline membrane which is secreted by the egg itself (b) chorion. the
intermediate layer secreted by follicular cells of the ovary and (c) the gelatinous,
tertiary egg membrane or albumen consisting of 3-4 gelatinous rings which is
secreted by the wall of the oviduct.
3. More than one half of the surface of ovum appears blackish due to the presence of
the melanin pigment while the rest appears almost whitish due to the presence of
yolk (Fig. 25.1).
-
4. The pigmented region will form the future animal hemisphere, while the non-
pigmented region will form the future vegetal hemisphere. Yolk
I
5. The cl& cytoplasm and haploid nucleus of the egg are located in the animal pole. Fig. 25,l : [Link].
6. The vegetal hemisphere contains the bulk of the yolk, which provide the necessary
nutrients for embryonic development.
SAQ 1
Can you explain the necessity for the three jelly coats in the ovum?
.- +
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Laboratoy Course n B Fertilized Egg
1. The centre of the animal and vegetal hemispheres are called animal and vegetal pole
respectively (Fig. 25.2).
2. The animal hemisphere contains the diploid nucleus formed by the union of sperm
and egg nucleus.
3. The vitelline membrane is now called fertilization membrane. Soon after fertilization
the egg secretes a protein material which absorbs water and swells up, causing the
vitelline membrane to lift up from the egg.
4. A grey crescent is visible opposite the point of entry of the sperm at the margin
between the animal and vegetal pole, largely in the animal pole. It is more lightly
pigmented than the animal hemisphere. The grey crescent later forms the posterior
and dorsal side of the embryo.
Fig. 25.2 : Fertilized egg. SAQ 2
With the help of labelled diagrams show the differences between the unfertilized and
fertilized frog eggs.

I C Blastula entire and V.S.

1. The blastula is a ball of cells with a central cavity the blastocoel (Fig. 25.3).
2. The section of blastula shows that it contains an excentric blastocoel cavity
surrounded by unequal sized blastomeres.
3. The smaller pigmented blastomeres are located in the animal hsmisphere and are
Blastocoel called microineres, while the larger yolk filled blastomeres occupying the vegetal
hemisphere are called megameres.
4. The micromeres at the animal hemisphere form the thin, multilayered roof over the
blastocoel, while the megameres of the vegetal hemisphere form the floot of the
blastocoel.
SAQ 3
Are all cells in the vegetal pole slightly larger, much larger or many times larger than
those at the animal pole?
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1

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Fig. 25.3 : Blastula SAQ 4
a) entire, b) V.S.
Draw a labelled diagram of the entire blastula and its vertical section.
D Gastrula entire and V.S. Study,ofPrepared SUdes of th 1
Developmental Stages 01 Fnby
The blastula gets transformed into a gastrula, which appears externally as a ball of dark
pigmented cells, with a circular, light coloured yolky plug.
1. The size of the gastrula is still as small as the zygote (Fig. 25.4a).
2. Gastrula is formed by rearrangement of cells of the blastula. Gastrulation -
reorganises the embryo compktely and results in three germ layers (1) external
ectoderm (2) The middle mesoderm and (3) innermost endoderm. The various . .: . . .
organs of the animal are &rived from these three layers. The various layers of rhe
gastrula are well differentiated and are clearly visible in vertical section (Fig. 25.4b).
3. The ectoderm will give rise to epidermis, cutaneous glands', nervous system, eye
I:,? . ' ;.:;!

parts and linking of mouth cavity and'cloaca.

4. Endoderm will form the lining of alimentary canal, liver, pancreas, lung urinary (a)
bladder and primordial germ cells.
5. The meso&rm~appears as a snlall area towards the posterior and will give rise to
the inusculature, connective tissue, vascular system, genital organs, excretory organs,
skeltton and notochord.
I
6. Other structures seen in the section are dorsal lip of blastopore, yolk plug, ventral
lip of the blastopore.
7. The notochord or chorda cells and neural plates are well differentiated and lie at the ....
dorsa! side.
8. The cavity of the gastrula called archenteron lies towards the dorsolateral side of the ':%.:_..
anterior part of the embryo, and the archeneteron will develop into gut in the future.
E Neurula Cb)
1. The embryo is called neurula as the two neural plates of the gastrula join to form a
nr~nmmlt n m L olnnn tho Innnit*nAinol ovic [Fin 3C C\
Fig. 25.4 : Gastrula
(a) entire (b) VS. I
2. Externally the neural tube in the embryo appears as a thickened ridge along the
longitudinal axis.
3. In vertical section of the neurula, the neural tube which is of ectodermal origin lies
above the notochord.
4. 'The notochord, a rod-like structure arises from the mesoderm in the middorsal
region'and lies below the neural tube and above the archenteron. The notochord Notochord
runs along the longitudinal axis of the embryo. Mesoderm I
5. On either side of the neural tube and notochord the extensive mesodermal tissues
are seen.
6. The mesodermal tissue which flank the neural tube form the somite .mesoderm.
7. The lateral mesoderm a continuation of the somite mesoderm appears split into
(1) The splanchnic mesoderm which lies nearest to the gut endoderm and (2) the
somatic mesoderm which lies nearest to the endoderm.
8. A new fluid filled cavity the coelom is present between the splanchnic and somatic
.mesoderm.
Gut
F Tadpole Larva of Frog
1. The egg hatches into a free living herbivorous larva (Fig. 25.6).
2. The larva measures about 5-7 mm in length.
I
3. The body of the larva is differentiated into body proper and tail with fin. Archenteron

4. The larva contains mouth, sucker, external gills, rudiments of eye, olfactory pits, Fig. 25.5 : VS. of Neurula
gut, anus and myotomes (muscles).
5. Mouth bears horny jaws or teeth.
6. Three pairs of external feathery gills act as functional respiratory organs.
7. Tail is long and bears the tail fin on its ventral and dorsal surface.
123
 The herbivorous larva feeds on vegetation and has a coiled intestine. The larva
metamorphoses into an adult.
T.S. of Tadpole through Eye Region
Eyes develop very early and are of ectodermal origin. They are the most
conspicuous and protuberant structures of the tadpole.
Eyes develop from the optic vesicles which originate from a pair of diverticula
given off from the prosencephalon of the brain on each side.
,Retina
Trabecula

' Palatoquadrat
bar
Sucker
Mouq 1

Jaw muscles - connective tissue

Fig. 25.7 : T S of tadpole through eye region.

Transverse section of the tadpole through the eye region (Fig. 25.7) shows the
forebrain in the centre on the doral surface.
The eyes are seen on the lateral sides of the brain and are composed of lens.
sensory layer of retina and eye muscles.
The pharynx lies in the centre of the transverse section.
Hyoid arch lies just below the phanynx in the section.
The palatoquardate bars are placed dorsal to the phaynx on the lateral sides.
Meckel's cartilage are present on the lateral sides of the pharynx.
Other structures seen in the section are jaw muscles, trabaculae, epidermis and
connective tissue.
T.S. of Tadpole through the Audltory Vesicles (ears)
Ears develop as a pair of auditory pits from the epiblast on the sides of the hind
brain.
Fig. 25.6 : Tadpole of frog The auditory vesicles develop at an early tadpole stage and are found on the dorso
lateral side of the section, below the hind brain (Fig. 25.8).

ourth Ventricle

Branchial arch

Second branchial

branchial arch .
/ / ~eri'dardial \ Fourth branchial
Auricle Ventricle Cavity Pericardium ar~h

Fig. 25.8 : T.S. of tadpole through auditory vesicles.

The auditory pits become vesicle like and enclose the middle ear. The semikrcular
canals develop as outgrowths from the wall of the auditory vesicles.
,The medulla oblongata of the hind brain is located in the middorsal region of the
section between the two auditory capsules and encloses the fourth ventricle.
5. The notochord lies below the medulla oblongata. Study of Prepared SUdes d the
Developmental Stages of Frog
6. The branchial chamber having the laryngeal chamber lies below the notochord and
auditory .vesicles.
7. The dorsal aorta and the first, second, third and fourth branchial arches are located
on the lateral side of the section.
8. The pericardial cavity enclosing the developing auricle and ventricle is located on
the ventral side of the section, below rhe laryngeal chamber.
I T.S. of tadpole through Head and Gills
Transverse section of the tadpole (Fig. 25.9) through head and gills shows the following
details.
1. The medulla oblongata enclosing the fourth ventricle lies in the middorsal position.
The membranous labyrinth are present on both the lateral sides of the medulla
oblongata.
The notochord lies just below the medulla oblongata.
The pharynx lies in the cenme of the section.
Larynx is seen in continuation with pharynx.
The heart enclosed in the pericardium is seen just below the larynx.
The branchial arches and internal gills are present on the lateral sides of the
pericardium.
The lateral dorsal aorta is located above the pharynx.
Lymphatic spaces lie on both the lateral sides.

Medulla oblongata

. Membranous
Labyrinth
Lateral dorsal

Pericardial Carity kart

Fig. 25.9 : TS. of Tadpole through the head and gills.

SAQ 5
1. Fill in the blanks:
a) A hollow ball stage in early embryonic development, preceding gastrula stage is
called a
b) The frog egg is called mesolecithal as it has amount of yolk.
c) A solid ball of cells in embryonic development is called
d) The cavity which forms during embryonic development and gives rise to the gut in
the adult is called
e) The coelom is a fluid filled cavity that separates the mesoderm from the
mesoderm.
d
f) Cleavage is but slow in the hemisphere due to the presence of
yolk in frog development.
Laboratory Cuurse II g) is the opening in the gastrulla through which the ectodermal cells
invaginate.
h) is infolding of cells through the blastopore'leading to differentiation of
endoderm and mesoderm.
2. Ultimately what is the fate of the blastocoel?

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3. From which germ layer does the nervous system develop?

4. Our study of the early developmental stages of frog has shown the formation of
three germ layers from which the major organs develop. In the table below indicate
the germ layers and their derivatives as discussed in this lab exercise.

Germ layer Organs developed

5. How does the size of blastula compare with one cell stage? These are about 1000
cells by late blastula stage in frog development. How many division would this
represent. What assumption is required for such a calculation? Are all the cells
similar in the blastula?

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6. Draw clear labelled diagram of all the stages you have observed.