1

VETERINARY EMBRYOLOGY

Exercise 4
Embryology

THE 18-hour CHICK EMBRYO

(whole mount)

The chick embryo is a blastula at the time of laying. After the egg is laid, development
stops until the temperature of the eggs is raised to 40oC (100oF) or nearly up the body temperature of
the mother. This temperature is reached when the hen sits on her eggs. Cooling of the egg does not,
however, result in the death of the embryo. It may resume its development if it is brooded by the
hen or artificially incubated even after the egg has been kept for many days at ordinary temperatures.
In the laboratory, the incubator takes her place. Developing eggs must be turned every 4 or 5 hours,
and a mechanical egg turner substitutes for the hen.
Incubation
The normal incubation temperature is that at which the egg is maintained by the body heat
from the brood-hen. This is somewhat below the blood heat of the hen (106oF). When an egg is
allowed to remain undisturbed, the yolk rotates so that the developing embryo lies uppermost. Its
position is then such that it gets the full benefit of the warmth of the mother.
In artificially incubating eggs, the incubators are usually regulated for a heat of 100o-101oF (37o-
38oC). In this temperature, the chick is ready for hatching on the 21st day. Development will go on
at considerably lower temperatures but its rate is retarded in proportion to the lowering of the
temperature. Below about 21oC, development ceases altogether.
In incubating eggs which have been cooled after laying for some particular stage of the
embryo which it is desired to secure, 3 or 4 hours are ordinarily allowed for the egg to become
warmed to the point at which development begins again. For example if an embryo of 24-hours
incubation age is desired the egg should be allowed to remain in the incubator about 27 hours. Even
with allowance* made for the warming of the egg and with exact regulation of the temperature of
the incubator, the stage of development attained in a given incubation time will vary widely in
different eggs. The factor of individual variability which must always be reckoned with in
developmental processes, undoubtedly accounts for some of the variation. The different time
occupied by different eggs in traversing the oviduct, the over-night retention of eggs not ready for
laying till toward sundown, and especially the varying time different eggs have been brooded before
being removed from the nest, account for further variations. The designation of the age of chicks in
hours of incubation is, therefore, not exact, but merely a convenient approximation of the average
condition reached in that incubation time.

Many of the developmental changes in the chick embryo during its first 96 hours (after
incubation begins) are almost identical with the events occurring in other vertebrates including
mammals. By the end of the 4th day of incubation, the embryo has all organs in miniaturized form.
 2

At this stage, the chick embryo is nearly indistinguishable from the mammal embryo at a similar
stage. After the 4th day, the characteristics specific to an avian begin to appear.

In 1951, Hamburger and Hamilton established a standard table of stages of chick

development. We will use a version of the HH staging table in this lab. A staging table divides
embryonic development based on observable features. The time (age) at each stage is an average
among many embryos since the timing of development is variable. A key feature of staging is the
regular (clocklike) addition of somites to the embryo in an anterior-posterior sequence.
Chick Gastrulation
The primitive streak is the major structural characteristic of reptile, bird and mammal
gastrulation. The streak begins to form at the posterior end of the epiblast (the outer layer of
blastomeres). As the primitive streak enlarges anteriorly a depression, the primitive groove, forms
within it. The anterior end of the streak is thicker and deeper than the remainder of the streak. This
thick area is called the primitive knot or Hensen's node. The depression in this area is called the
primitive pit. From the epiblast, the endoderm and mesoderm ingress through the primitive groove
or primitive pit.
Endoderm Formation. The first cells which ingress are the prospective endodermal cells.
As the endodermal cells enter the blastocoel, they migrate anteriorly and laterally. The migrating
endoderm pushes the hypoblast out of the way, and forms the deepest layer of the embryo. The cells
destined to become the pharyngeal endoderm ingress through the primitive knot.
Mesoderm Formation. The second wave of ingressing cells forms the mesoderm layer.
These mesodermal cells spread between the endoderm and the overlying epiblast. As the mesoderm
is ingressing, the primitive streak has reached its maximum length. The prospective mesoderm
which enters the primitive knot moves anteriorly to form the head process. The head process forms
head mesenchyme and the anterior end of the notochord. Posteriorly to the primitive knot the
ingressing mesoderm spreads laterally and anteriorly.
Regression of the Primitive Streak. While mesodermal ingression continues, the primitive
streak starts to regress. The primitive knot moves posteriorly. As the knot moves posteriorly, the
notochord is laid down, starting at the level of the future mesencephalon and moving toward the anal
region. As a consequence, the embryo exhibits a distinct anterior-posterior gradient of
developmental maturity. As the cells at the anterior end of the embryo are forming organs, cells at
the posterior portion of the embryo are continuing to ingress.
Ectoderm Formation. As the prospective endoderm and mesoderm enter the primitive
streak, the ectoderm spreads to cover the vacated area on the surface of the embryo.

ACTIVITY
 3

Examine a whole mount of an 18-hour chick embryo under the scanner (4x) and refer to the
diagram in Patten’s Early Embryology of the Chick (Figure 1 below) or the Patten’s embryology by
Carlson for your guidance.

Fig 1. Dorsal view of and 18 hour chick embryo.

By 18-hours of incubation, the primitive streak has been fully established and the embryo
enters the 2nd and 3rd stages of gastrulation which are the establishment of the primary germ layers
and the formation of notochord, respectively.
There are three primary germ layers, namely endoderm, mesoderm, and ectoderm. However,
the positional relationship of these germ layers is not demonstrable in the whole mount. Like the 16-
hour chick embryo, the whole mount of an 18-hour chick embryo presents the following structures:
primitive streak with primitive groove and primitive ridge, Hensen’s node and primitive pit, area
pellucida, area opaca and embryonal area. In addition to these structures, a notochord, a proamnion
and a neural plate are the new structures present in the 18-hour whole mount.
The notochord appears as an elongated structure extending caphalad from the Hensen’s node,
in the midline of the area pellucida. Locate the darker stained neural plate on either side of the
notochord. The neural plate is derived from the ectoderm and is the earliest indication of a
developing nervous system. In the whole mount, the notochord appears as though it lies in the
middle of the neural plate but it does not. The notochord actually lies beneath the neural and only
appears distinct because of the transparency of the neural plate at this stage.
Anterior to the notochord and the cephalic end of the area pellucida,observe a crescent
shaped light area, the proamnion (before the amnion), which is the portion of the area pellucida not
 4

yet reached by the mesoderm. The light appearance of the proamnion is therefore due to absence of
mesoderm beneath the ectoderm in this area.

Whole Mount of an 18-hour Chick Embryo

Identify the parts (indicated by numbers) of the embryo (18-hour) depicted above. Use any textbook
or by just using Figure 1 above.
1. ____________________
2. ____________________
3. ____________________
4. ____________________
5. ____________________
6. ____________________
7. ____________________
8. ____________________

Below is picture of a 20-hour chick embryo. Identify its parts and then compare these to those seen
in an 18 hour chick embryo. Did you find any difference? If yes, what are these?
________________________________________________________________________________
_______________________________________________________________________________.
 5

1. What structures give rise to the three primary germ layers? _______________________
2. What structure forms the
a. Roof of the primitive gut? _________________
b. Floor of the primitive gut? ________________
3. How does the proamnion differ from the area opaca?
__________________________________________________________________________
__________________________________________________________________________
___________________________________________________________________.
4. What is the significance of the notochord in a developing vertebrate embryo?
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________.
5. How do you differentiate a 16-hour chick embryo from an 18-hour chick embryo?
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________.