THE CHICK EMBRYO AS A MODEL FOR STUDYING BRAIN DEVELOPMENT,

FUNCTION, AND TISSUE SCAFFOLD DEVELOPMENT

Balagot, K.M.DR. and Tagudando,R.M.B.
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand

ABSTRACT

The chicken embryo as a model has a lot of advantages in research. It is convenient to be studied
for its short developmental time; and it is observable in ovo. This experiment aims to utilize
chicken embryo as a model for studying the development and functions of the brain, and for
developing tissue scaffolds. Incubation and egg windowing are first performed prior to the
observation-proper. Other techniques that are essential in this study are: Choroid Plexus Isolation,
RT-PCR, staining procedures (H and E, ORO, ICC, IHC), CAM Assay, and scaffolding.

A. BRAIN DEVELOPMENT

The brain is one of the largest and complex organ in the human body. It is the head of the nervous
system due to its billion nerve cells that communicate through synapses to send signals all over
the body. Its development start from the thickening of the ectoderm that later on forms the neural
plate and the neural fold or crest. Once the neural plate folds longitudinally and the folds or crest
fuse together it will form the neural tube which will then be simultaneously separated from the
ectoderm so that its cephalic portion can be enlarged to form the brain. The brain has three
primary vesicles: prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon
(hindbrain). The rhombencephalon will develop first and it will be divided into two:
myelencephalon and metencephalon. This division will lead to the bending of the brain that
displaces prosencephalon enlarging its cephalic portion and soon enough will be divided into two:
telencephalon and diencephalon. The five secondary vesicles of the brain each has their own
significance to protect and assist in the brain function. The telencephalon contains the foramen
of monro that is responsible for transporting the cerebrospinal fluid from the lateral ventricles to
the third ventricle. It will then be divided further into two: cerebrum and lateral ventricles. The
diencephalon maintained its thin wall so that ingrowth of blood vessel could still be possible and
be pushed towards the third ventricle in order to form the anterior choroid plexus. The
mesencephalon contains the aqueduct of sylvius which is also responsible for the transport of
cerebrospinal fluid from the third ventricle down to the fourth ventricle. The metencephalon divides
more into three: cerebellum, medulla, and pons. The myelencephalon produces blood vessels
which is then pushed towards the myocoele that forms the posterior choroid plexus.

B. CHOROID PLEXUS DEVELOPMENT

The choroid plexus is a structure with a highly vascularized tissue; this has a crucial role in forming
one of the blood-brain barrier interfaces. There are four choroid plexuses in human brains as well
as in the chicken embryo brains. There are two choroid plexuses that appear in the lateral
ventricles of the telencephalon, one in the third ventricle of the diencephalon, and one in the fourth
ventricle of the hindbrain. Choroid plexuses develop from several locations along the dorsal axis
of the neural tube, after the neural tube closure. In humans, the hindbrain choroid plexus (hChP)
of the fourth ventricle is the first one to appear, this is followed by the development of the
telencephalic choroid plexuses in each of the lateral ventricle, the last to show up is the
diencephalic choroid plexus of the third ventricle—but this is also the ChP to finish differentiation
the earliest. However, in the case of the chicken embryo’s brain: telencephalic choroid plexus
starts to develop first. The morphological development of the choroid plexus is divided into four
stages, at first the choroid plexus appears to have pseudo-stratified epithelial layer with centrally
located nuclei in stage 1—with no villi. In stage 2, transition to a columnar epithelium with apically
located nuclei proceeds, and is followed by the formation of primary villi and convoluted tissue. In
the third stage, it appears that the epithelial cells flattened becoming cuboidal cells, nuclei are
centrally or apically located, and villi in the tissue become more abundant. Lastly, nuclei become
more basally located, this is because of the apical enrichment of transport machinery and
microvilli, and villi are more complex with multiple fronds. Junctions and adhesion molecules are
found between adjacent cells, allowing the mature choroid plexus to function as the blood–
cerebrospinal fluid (CSF) barrier by restricting the passage of solutes from the systemic circulation
into the CSF.

C. CEREBROSPINAL FLUID

Cerebrospinal fluid is composed of 99% water and the remaining 1% divided into ions, hormones,
growth factors, nutrients, and biomolecules such as lipids and proteins. It keeps the brain
stabilized with in the skull and it provides cushion for shock absorption. It also functions as a
transporter so that the whole brain can be supplied with the nutrients it needs. The flow of the
cerebrospinal fluid starts from the lateral ventricles down to the foramen of monro to the third
ventricle down to the aqueduct of sylvius to the fourth ventricle and passing through the arachnoid
spaces so that the entire brain can accumulate every nutrient present in the cerebrospinal fluid.

D. CHICKEN EMBRYO AS MODEL

Chicken embryo has crucial roles as models in drug testing, retinal development, embryonic
development and more experiments in research. In this experiment, chicken embryo is utilized as
a model for studying embryonic and brain development. This is because not only of the 60 %
similarity of the avian genes to humans, but also: because it matures for a short span of time, its
changes during development could be easily observed in vivo and under a microscope.

E. INCUBATION

One of the first techniques that is tackled prior to and during observation of the embryonic
development is the incubation of the chicken embryo—chicken embryo egg. Incubation is defined
as the development of embryo in the egg, whereas incubator—which is the primary equipment n
incubation—is utilized to serve as an environment with conditions required for the embryo to
develop. In incubation, there are three three significant factors to be carefully considered, these
are the: temperature, humidity, and ventilation. For the temperature: The eggs need to be kept at
99.5 degrees at all times; just one degree higher or lower for a few hours can terminate the
embryo. For the ventilation: Egg shells are porous, allowing oxygen to enter and carbon dioxide
to exit; incubators need to have holes or vents that allow fresh air to circulate so the embryo can
breathe. Lastly, for the humidity: 40 to 50 percent humidity must be maintained for the first 18
days; 65 to 75 percent humidity is needed for the final days before hatching. Unnecessary loss of
egg moisture must be prevented.
 F. EGG WINDOWING

Egg windowing is a technique used to observe the properties of the chick embryo better. It is
usually done after three days of incubation so that the egg shell is strong enough and the blood
vessels are not yet attached to the egg shell.

MATERIALS

Fertilized egg, scotch tape, scissors, and syringe with needle

METHODOLOGY

Poke the egg at the bottom where in the needle of the syringe can fit. Hold the egg
horizontally and place the needle in a perpendicular position. Remove about three
milliliters of albumin so that the embryo would go down to prevent the blood vessels to
attach to the egg shell. Place a tape on top of the area of the egg shell desired to cut so
that the other areas would not be damaged. Cut a circular portion big enough to view the
whole chick embryo. Afterwards place a tape to seal to prevent contaminations.

G. ISOLATION OF THE CHOROID PLEXUS

One of the safest technique for the isolation of the choroid plexus is by first, understanding the
anatomy of the brain. Primarily, when viewed dorsally or ventrally, the brain has three major
parts—the forebrain, midbrain, and hindbrain.

METHODOLOGY

After the anatomical familiarization, the midbrain is poked with forceps, this is to hold the
brain when separating the forebrain and hindbrain from the midbrain. After the separation,
forebrain and hindbrain are slowly lacerated in cross-section to obtain the choroid plexus.

Figure 1. Above are the forceps utilized for performing the isolation. The middle picture shows the dorsal view of a
brain. The last picture shows the midbrain poked with forceps.
RESULTS

Figure 2. (A) The lateral choroid plexus appears to have a branch-like feature, (B) while the third ventricle choroid
plexus is like a corral, (C) while the hindbrain choroid plexus of the fourth ventricle has a wing-like structure.

H. REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR)

RTPCR is a process of amplifying DNA starting with a RNA template. The cycle goes from
initiation by using a complementary primer to bind with the RNA strand and a polymerase to start
elongation to create a cDNA. In order to make a double stranded DNA, another set of
complementary primers attach to the strand and a polymerase to elongate. The cycle continues
until the desired number of amplicons are reached.

MATERIALS

RNA from an E20 brain, primers, PCR-grade water, HotStart ready mix, PCR tubes,
micropipettes, and thermocycler

METHODOLOGY

Prepare eight PCR tubes and place each about 8µL of PCR-grade water, 12µL HotStart
ready mix, 10µL of forward primer, 10µL of reverse primer, and 4µL of the template.
Ensure that the caps are tightly closed and place them in the thermocycler. Input the
required conditions and view it the next day using agarose gel electrophoresis.

RESULTS

Unfortunately, after repeating the experiment twice, the researchers only observed the
bands coming from the DNA ladder. As a conclusion, the template must have been
degraded over time. However, a senior provided us result for this experiment. In the right
side of the figure below, it can be seen that bands are present expressing the following
 genes responsible for fatty acid and cholesterol synthesis not just in the liver but also in
the brain. Therefore, this experiment can be used to provide evidence that the brain can
synthesize its own lipid and fatty acid content specifically the choroid plexus.

Figure 3. The experimental results from the researchers that only shows the DNA ladder (left). The results from a
senior that depicts the expression of genes by the brain responsible for fatty acid and cholesterol synthesis (right).

I. OIL RED O STAINING

Another technique or the confirmation of the lipid content of the choroid plexus is the Oil Red O
(ORO) Staining. ORO is used to demonstrate the presence of fat or lipids in fresh, frozen tissue
sections. It is an oil-soluble pigment, wherein it appears to be in red color when viewed under
microscope. The more the red color becomes evident means that there’s a great amount of lipid
in that particular area. Aside from its applications in research, ORO could e also used in:
Diagnostic labs, pathologists use ORO to help diagnose various conditions in which fat may
appear in abnormal locations. For instance, bone fractures or crush injuries to fatty regions of the
body may release fat into the bloodstream, leading to fat emboli which can be fatal- these emboli
are detected using ORO. It is also useful to identify tumors, such as lipomas and liposarcomas,
which arise from fat cells. Deposits of fat may also appear in the liver and kidney in a variety of
pathological conditions. It is also used in forensic pathology as a reagent for fingerprint
development. Detecting fingerprints on porous surfaces which have been exposed to moisture is
difficult as the amino acids from the prints will dissolve in water. ORO as a reagent to enhance
latent prints produced by the lipids found in fingerprints

METHODOLOGY

For the preparation of the ORO stain, this is done by mixing 500 mg of ORO powder into
100 mL of isopropanol. After preparation, few drops of ORO stain were put on the cross-
section tissues of the brain—with the choroid plexus.
 RESULTS

Figure 5. The photos above show the darker red coloration of the ependymal and choroid plexus compared to the brain
parenchyma.

J. FIXATION

The brain will begin to deteriorate as soon as its blood supply is interrupted or it once it is isolated.
The deterioration is rapid, and it is, therefore, important to arrest the deterioration as quickly as
possible. Cells are permeated to allow antibodies to access intracellular structures. Without
fixation, the structures in cells would fall apart and diffuse away. Some of the commonly used
fixative solution are the paraformaldehyde, alcohol, and the gulataraldehyde. In our experiment,
paraformaldehyde is used for brain’s fixation. Basically, paraformaldehyde causes covalent cross-
links between molecules, effectively gluing them together into an insoluble meshwork.

K. CRYOSECTIONING

The most significant and first technique for the observation of a brain tissue is performing the
cryosection. Cryosectioning is a method of cutting a sample into thin sheets—micrometer tissue
samples, without damaging the tissue. Some of the major equipment that are encountered are
the cryostat and theOCT (optimal cooling temperature), OCT is a solution that is viscous at room
temperature and miscible with H2O, but freezes into a solid support at −21°C. This holds and keep
the sample off from breaking when cut through the cryostat. Thus, the technique cryosectioning
provides a good system for visualizing fine details of the cell. Because of its rapid freezing, it
reduces ice crystal formation and minimizes morphological damage. Frozen sections cut in here
may be used for a variety of procedures, including immunochemistry, enzymatic detection, and in
situ hybridization.
 L. CHORIOALLANTOIC MEMBRANE (CAM) ASSAY

CAM is a membrane formed by the fusion of chorion and allantois. It is a covering of the chick
embryo that is rich in vascular networks providing an interface for gas and water exchange. Its
cellular matrix is made up of laminin, fibronectin, collagen, and glycosaminoglycans. Researchers
mostly use this technique to study for tumor growth, drug delivery, and angiogenic molecules.

M. SCAFFOLDING

A scaffold is basically a framework that provides a connection between two things. The scaffold
material is made from silk fibroin from a worm called Bombyx Mori. It is used for it has the same
components found inside the CAM. This is an important method to enhance tissue repair and
regeneration.

MATERIALS

Scaffold, Cryosection, and CAM of chick embryo

METHODOLOGY

Place the scaffold in the CAM. Observe for several days of incubation. Afterwards, cut the CAM
with the scaffold. Score the scaffold on how much blood vessels penetrated into it. Ready for
cryosectioning, cut into 40 microns and view under the microscope.

N. HEMATOXYLIN AND EOSIN (H&E) STAINING

H&E staining is used to view the morphological properties of the cell. It differentiates the nucleus
from the cytoplasm. Hematoxylin should be oxidized to form the active ingredient in staining which
is hematoin. Hematoin bound with the metal ion alum binds to the phosphate group of the
nucleotide found in the nucleus and that gives a blue coloration. On the other hand, eosin is an
acid stain that binds to the basic amino acids found in the protein of the cytoplasm and it gives a
pink-red coloration.

MATERIALS

Tissue sample, EtOH (different percentage), hematoxylin, HCl, eosin, and tap water

METHODOLOGY

Cells need to be hydrated before staining so that the stain can penetrate more in the tissues.
Place the slides on increasing percentage of alcohol for 30 seconds each. Then place on the
hematoxylin stain for about 3 minutes. Wash with tap water and drop HCl to further remove excess
stain. Place in the eosin stain for 10 minutes and wash with tap water then directly place into
decreasing percentage of alcohol to dehydrate and remove water contents.
RESULTS

Figure 5. After H&E staining the nucleus and the cytoplasm of the tissue was not distinctly differentiated.

The picture above hardly shows the difference between the nucleus and the cytoplasm based on
its color. To conclude, there might be a problem in the staining procedure or in the stains itself.

O. IMMUNOHISTOCHEMISTRY

Another way to observe brain sections is by using immunohistochemistry (IHC) staining. IHC is a
method to detect specific antigens in cells based on an antigen-antibody reaction which can be
recognized at the light microscopic level. This is prepared by mixing 50 mM TRIS buffered saline

and adding 1 % Triton X-100 to a proportion of the solution– for lysing the cells. The factors to
consider in performing IHC are the following: Fixation, Sectioning, Labeling, Blocking, and
Antigen Retrieval. Moreover, immunohistochemistry might have quite similarities with
immunocytochemistry (ICC), however these techniques differ with sample source and sample
processing: (a) IHC uses tissue sections, either paraffin embedded or frozen, whereas ICC refers
to the staining of isolated or cultured intact cells. (b) ICC samples, compared to IHC, undergo a
shorter fixation period. Also, an antigen retrieval method is typically included before commencing
IHC staining to restore tissue antigenicity.

P. IMMUNOCYTOCHEMISTRY

Immunocytochemistry is a staining procedure involving antibodies. It has four main steps:

seeding, staining, visualization, and analysis. Seeding is done with incubation to ensure that the
cells are attached to the sold support. Before staining, the cells must be fixated to preserve the
characteristics of each protein and permealized to remove the lipids so that the antibodies can
easily pass through the membrane. The microscope is used to view the stain and later on
analyzed.

Q. EFFECT OF ALCOHOL ON CHICK EMBRYO

Sometimes pregnant women intake alcohol without knowing the risks of their baby having
abnormalities. This study is to test on how much can alcohol affect the embryo.
 MATERIALS

Fertilized egg, 10% alcohol, and 15% alcohol

METHODOLOGY

Drops of alcohol were placed into an embryo and incubated so that it can be observed
after several days.

RESULTS

Figure 6. Shows the brain and the choroid plexus from an E12 chick embryo from the control, to the ones treated with
10% and 15% alcohol.

At first glance, the differences among the three are not easily noticed, but when you look closely,
some parts of the brain treated with alcohol swelled and shrank and the choroid plexuses broke
and swelled. For this, it can be concluded that in taking alcohol while pregnant can cause severe
brain damages that can affect almost all parts of the body. When the brain, the control center of
humans is malfunctioned the transport of signals throughout the body will be affected. As well as
when the choroid plexus is damaged there would be no producer of cerebrospinal fluid that
contains the nutrients for the brain.
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types-applications-and-animation/
■ Deathful Follow. (2014, January 11). Egg Windowing. Retrieved July 2, 2017, from
https://www.slideshare.net/CNuggets/egg-windowing
■ Lovett, M. L., Cannizzaro, C., Daheron, L., Messmer, B., Vunjak-Novakovic, G., & Kaplan,
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