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LABORATORY EXERCISE – 4 mm FROG EMBRYO

Transverse Section
Locate the most anterior sections in the series and identify the following structures as
you examine the series progressively toward the posterior.

An oval group of head ectoderm cells which enclose another group of brain ectoderm
cells, with an innermost cavity known as the prosocoel (cavity of the forebrain or
prosencephalon). Ventrolaterally locate two pigmented invaginations of head ectoderm, on
either side of the prosocoel, representing the very beginnings of the olfactory pits. In the
midventral position locate the dorsally directed invagination of this head ectoderm which is the
beginning of the stomodeal cleft, an ectodermal invagination which will give rise to the lining of
the mouth. Between the brain and the head ectoderm identify the scattered embryonic
mesoderm cells known as head mesenchyme. In this vicinity, locate also a slight middorsal
evagination of the brain vesicle known as epiphysis, a structure which becomes the pineal body
in the adult of many vertebrate forms.

Several sections more posteriorly note that the nasal pits have disappeared entirely, as
have also the stomodeal cleft and epiphysis. This will convey the impression of the relative sizes
of these structures. The brain cavity is now much larger and is known as mesocoel or cavity of
the thick-roofed mid-brain (mesencephalon). Near the position of the original stomodeum there
appears a triangular space surrounded by the large but unpigmented cells which is the pharynx
which will later join the invaginating ectodermally lined stomodeum to form the mouth.

The ectodermal hypophysis is directly dorsal to the pharynx lying between the
pharyngeal and brain cavities. The larger more anterior ventral to the brain cavities is the
prosocoel with thin walls. Two lateral evaginations of the prosocoel known as opticoels because
they will give rise to portions of the optico-ocular apparatus. The lateral walls of these
opistocoels are much thicker than the adjacent walls of the brain. In later stages these lateral
optic evagination will begin to evaginate to form the optic cups. The most lateral and thick wall
of the original opticoel will become the retinal portion of the eye. As the dorsal margin of this
optic cup comes into contact with the superficial ectoderm it induces the development of the
lens placode.

The most ventral portion of this optic cup does not come into contact with the outer
ectoderm but remains to form inverted groove known as the choroid fissure. The connection of
the optic cup with the brain is the optic stalk. The surrounding loose head mesenchyme is easily
identified, which will give rise to the connective tissue and skeletal structures in the region of the
eye.

In more posterior sections note that the dorsal brain cavity is much reduced at the level
of the hindbrain or rhombencephalon. The cavity of the hindbrain is called rhombocoel and may
be distinguished by its thin roof. Lateral and slightly ventral to the brain locate paired
invaginations of the superficial ectoderm known as the auditory vesicles. Midway between these
vesicles is the well-formed notochord with its large vacuolated cells. Loose mesenchyme cells
are abundant which will give rise to the auditory capsule.

Lateral to the enlarged foregut locate the slit-like openings which are the beginnings of
the aortic arches, not yet supplied with blood corpuscles. Beneath this level of the foregut the
mesoderm is beginning to form the embryonic heart. Heart mesoderm migrates from the two
sides to this midventral position giving rise to large semicircular cavity which will become the
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pericardial cavity. The surrounding mesodermal membrane will become the pericardium. Within
the pericardium, you will see a much thicker mass of mesoderm which is the myocardium.
Scattered within the myocardial cavity are loose mesodermal cells which will soon become
organized into a lining as endocardium. At about this level, locate the two pigmented
ventrolateral ectodermal invaginations known as the oral suckers.

Examine the serial sections more posteriorly until the foregut is much reduced dorsally
but has a slit-like connection with a more ventral cavity, the liver primordium. It should be clear
that the foregut above and the liver below are lined with the same type of endodermal cells.
Surrounding the entire area is mass of undigested yolk. Ventro-laterally to the liver, at the
margin of the yolk and splanchnic mesoderm, locate the vitelline veins. In later stages numerous
vitelline veins will appear. The hypomeric mesoderm, consisting of this splanchnic layer plus the
outer somatic layer, surrounds the yolk mass except for the middorsal region. You can see that
the outer somatic mesoderm is in close contact with the body ectoderm – now called the
somatopleure. When the splanchnic mesoderm becomes associated with the gut endoderm, the
two layers will be the splanchnopleure.

Directly above the gut is the sub-notochordal rod and dorsal to that is the large
vacuolated cells of the notochord. On either side of the hypochordal rod is the paired dorsal
aortae. Lateral and ventral to the aortae the intermediate mesoderm (mesomere) is now
organized into circular groups of cells which will become the pronephric tubules.

Just lateral to the notochord find the epimeric mesoderm which is segmented into
somites. The brain cavity at this level is still the rhombocoel but the roof is much thinner than the
floor. From the junction of the neural tube and the outer ectoderm there will appear aggregates
of cells, lying dorso-laterally to the developing central nervous system. These are the neural
crests, the forerunners of the spinal ganglia, chromatophores and sympathetic nervous system.

Several serial sections further posteriorly, note that the liver cavity is separated from the
foregut; the pronephric tubules are better formed and the loose sclerotomal cells have broken
away from the somites to cluster around the notochord. These cells will give rise to the axial
skeleton. The major part of the somite will remain as the rather solid myotome while an outer
layer of cells will become closely associated with the outer ectoderm as the dermatome – to
form connective tissue and blood vessels of the skin.

Sections through the mid body level will show the same structures but size relations may
be different. The liver, heart and foregut will no longer visible.

I. QUESTIONS FOR RESEARCH:

1. What are the major parts of the heart at the 4mm stage? Relationships?

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2. What are the meanings of: visceral or branchial as adjectives; arches, pouches or clefts as
nouns?

II. ILLUSTRATIONS:
Draw a transverse section through the level of the opticoels and a section with reduced foregut
and liver primordium. Completely label the parts.

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Sagittal Section
The most medial sagittal section is the one which shows the maximum of the brain cavity
and the neurocoel. Note that the forebrain vesicle is flexed ventrally around the tip of the
notochord at the region of tuberculum posterius, and that the most anterior projection of the
forbrain is the epiphysis. In the floor of the prosencephalon locate the two thickenings, the
posterior optic chiasma and the anterior torus transversus, with the intervening depression
called the optic recess. At this time, it is possible to draw an artificial line between the anterior
telocoel and the diocoel (subdivisions of the prosencephalon) by locating the posterior limit of
the choroids plexus and the anterior limit of the optic recess. Posterior to the optic chiasma, the
diocoel bulges beneath the notochord as the infundibular cavity, the brain portion of which will
aid in the formation of the hypophysis. Posterior to the dorsal thickening and dorsal to the
notochord, locate the hindbrain which merges with the neurocoel.

In these sections it will be possible to determine the extent of the notochord. Its most
anterior tip is between the tuberculum posterius and the infundibulum, and the most posterior
limit is the developing tail. Notochordal cells are always easily distinguished because they are
large and vacuolated. The subnotochordal rod may be difficult to locate because it is too small.

The relationship of the hypophysis to the infundibulum (floor of the diocoel) and the roof
of the foregut (pharynx) will be clearly indicated since the hypophyseal cells are pigment and
grow between these regions. The heart, forms as a bilateral organ, then becomes a bent tube.
In these sagittal sections, parts of the heart may appear separate from each other, although all
will enclosed by the pericardial membrane. The thyroid is a single evagination form the floor of
the pharynx just anterior to the heart. The liver is a prominent diverticulum of the foregut which
projects postero-ventrally behind the heart.

At the posterior end, the anal opening should be located as a newly formed ectodermal
invagination (proctodeum) directly ventral to the position of the original blastopore. Both the
proctodeum and the remnant of the neurenteric canal may be identified by pigmented cells.

In the more lateral of the sagittal sections, locate the pronephric ducts; the auditory
vesicles; the optic stalk and more laterally choroid fissure; the segmental somites and in the
region of the pharynx, the lateral evaginations of the endoderm which will form the visceral
clefts. The most anterior pouch, later to form a cleft is the hyomandibular. This is followed by the
first to fifth gill pouches.

III. ILLUSTRATION:
Draw the most medial sagittal section of the 4mm tadpole. Completely label the parts.

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IV. QUESTIONS FOR RESEARCH:
1. What evidence are there of the original position of the blastopore?

2. How far anterior and posterior does the notochord extend?

3. Describe the blood vascular system of the 4 mm tadpole.

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Frontal Section
These frontal sections are mounted from the most dorsal to the most ventral so that the
first longitudinal cavity to be seen will be part of the central nervous system with posterior
neurocoel. The first organs to appear will be the epiphysis and lateral otic (auditory) vesicles.
Just anterior to these vesicles locate aggregations of neuroblasts representing the large 5th and
more posterior 7th and 8th cranial ganglia. The 7th and 8th arise together, later to be separated as
the facial and auditory.

In more ventral sections, at about the level of the notochord, locate the optic cups with
lenses, just anterior to the region of the 5th cranial ganglia. At this level the small 9th cranial
ganglia, and the 10th with posterior extensions, the lateral line nerves, may come into view. The
only visible at this level is the most anterior prosencephalon. Just posterior to the prosocoel,
where the notochord is disappearing, locate the most dorsal portion of the pharynx.

Locate next the olfactory placodes, lined with pigmented ectodermal cells; the optic
stalks and vesicles; the infundibulum and midgut and hindgut. On some slides it may be
possible to see the metamerically arranged spinal ganglia on either side of the notochord or
neurocoel. Within the visceral arches note the large empty blood vessels (aortic arches) some
of which will later grow out into the developing external gills as branchial vessels. The
hypophysis will be seen anterior to the pharyngeal cavity as a median column of pigmented
cells just ventral to the disappearing infundibulum as the more ventral sections are reached.

More ventrally the midgut disappears. Just posterior to the pharynx, the heart structures
will appear consisting of paired vitelline veins converging from the posterior; the endocardium,
myocardium, and pericardium. These are particularly good sections in which to determine the
changes in shape of the embryonic heart. Between the heart and the pharynx is the rather large
mass of darkly staining cells which represent the thyroid gland which later migrate posteriorly.
Posterior to the heart and embedded in the yolk, locate the rudiment of the bile duct leading into
the ventral liver diverticulum. At the posterior end of these sections note the pigmented
proctodeum. Still more ventrally, at the anterior end, find the oral suckers.

V. ILLUSTRATION:
Draw the frontal section of the 4mm tadpole (at the level of the sense organs and cranial
ganglia). Completely label the parts.

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VI. QUESTIONS FOR RESEARCH:
1. In relation, to the notochord, at what level do we find the pronephric ducts in the frontal
sections?

2. Compare the following organs in transverse, sagittal and frontal sections:

a) thyroid gland c) liver diverticulum
b) infundibulum d) optic stalk and vesicle