1. “Immunohistochemical localization of Nitric
Oxide Synthase (NOS) during early development
of the chick embryo”
Guide
Asiya N. Khan Dr.Shobha Bhargava
2006-ZY-10
Department of Zoology
University of Pune.

2. Introduction to “Nitric oxide synthase
(NOS)”
• NOS is a 150-kDa protein.
• It is an enzyme which acts in a NADPH-dependent manner.
• It was first identified by Furchgott in the aortas of rabbits.
• require five bound cofactors/prosthetic groups: FAD, FMN, haem,
tetrahydrobiopterin (BH4) and Ca2+-calmodulin (CaM).
• NOS is competitively inhibited by the NADPH-d substrate, nitro blue
tetrazolium

3. Isoforms of NOS
• Neuronal NOS (nNOS or NOS1)
- Always present in the cells
- soluble and found predominantly in the cytosol
- role in cell communication
• Inducible NOS (iNOS or NOS2)
- produced when needed.
- soluble and found predominantly in the cytosol
- immune defence against pathogens.
• Endothelial NOS (eNOS or NOS3 or Constitutive / cNOS)
- Always present in the cells
- membrane associated
- regulating vascular function.

4. Summary of the functions of NOS
• Transmission of impulse from one neuron to another .
• Immune system .
• Dilating blood vessels

5. Reaction mechanism of NOS
• • NOS produces nitric oxide (NO)
by catalysing a five-electron
oxidation of a guanidino
nitrogen of L-arginine (L-Arg).
• Oxidation of L-Arg to L-
citrulline occurs via two
successive monooxygenation
reactions producing NOHLA as
an intermediate.
•Two mol of O2 and 1.5 mol of
NADPH are consumed per mole
of NO formed.

6. Nitric oxide (NO)
• Nitric oxide (NO) is a membrane-permeable gas that was first
identified as endothelium-derived relaxing factor.
• It is synthesized in the nervous system and functions as an
important intercellular messenger in neurotransmission,
excitotoxicity, plasticity, and control of cerebral blood flow
• At low concentrations NO functions as a signal in many diverse
physiological processes such as blood pressure control,
neurotransmission, learning and memory, and at high
concentrations as a defensive cytotoxin.

7. Facts about NOS & NO in Chick
It is reported that high levels of NOS is present in the early stages of
developing chick embryo retina.
It is also reported that there is an increase in the expression levels of NOS
in the visual structures of the chick brain after retinal removal.
It is also seen that the expression of NOS in the optic tectum of developing
chicks is regulated by the retinal innervation.
In chickens, the elimination of the ipsilateral retinotectal projections is
partially dependent on the NO synthesized in the tectum opticum
NO is synthesized in the chick embryo retina and brain prior to
synaptogenesis indicating that NO production may be considered as a
signal for the development and shaping of neuronal and non-neuronal cells

8. However, there are no reports on the occurrence or role of
NOS during the early development (gastrulation & neurulation)
of Chick embryo.
Herein, we report for the first time the localization of NOS
during the early development in chick.

9. Animals and tissue preparations:
• Freshly fertilized white leghorn chicken eggs were collected from IBVP
poultry, farm and were incubated at 37oC.
• Embryos were harvested by the filter paper ring technique and were then
washed in 0.9% saline
• Fixed in Bouin’s fixative for 24 hrs.
• Embryos were then dehydrated by passing through different graded
alcohol.
• Embryos were then serially sectioned (7µm) in transverse plane.
• Mounted on the slides coated with the Poly-L-Lysine.
• These slides were stored and then used for the immunohistochemical
reaction.

10. IMMUNOHISTOCHEMICAL PROTOCOL
Sections were deparaffinized
Sections were then hydrated
treated with 0.3% H2O2 in methanol
washed in PBS-I (20 min)
incubated in BSA gelatin
washed in PBS-I (10 min)
incubated with diluted (1:40) goat serum
incubate with primary antibody (overnight)

11. washed in PBS-I (10 min)
Incubate with biotinlyated secondary antibody
washed in PBS-I (10 min)
Incubate with ABC
washed in PBS-I (10 min x 3)
section were reacted in dark with DAB
washed in tap water
Dehydrated, cleared and mounted in DPX.

12. Results
Primitive streak Stage 4
• Following 18 hrs of incubation (stage-4) very strong NOS immunoreaction
was observed in the area pellucida.
• In the area pellucida just before the beginning of primitive streak positive
NOS reaction was observed.

13. • In
the anterior most part of the primitive streak a very strong immunopositive
reaction was observed (Fig. A, B)
eec
m
ee
A a m
eec
Pg ps
m
np
Pg ps
yp
B B

14. • In the middle region of the embryo the NOS positive reaction was also
observed in the embryonic ectoderm, mesoblast, neural plate, while in the
embryonic endoderm the reaction was negative (Fig. A,a).
ps
Pg
np
eec
ps m
a
Pg
np
m
A ee
• At the posterior most part of the embryo, the reaction was similar to
that of the anterior part but weak reaction was also observed in the
embryonic endoderm (Fig. B,b).
eec
np
np
ee
B b

15. • During neurulation at stage-10 when the neural fold starts fusing, the
overall immunoreaction was strong.
• In the forebrain region and mid brain region the reaction was seen in the
cell of lateral part of the neural tube.

16. •The positive reaction was observed in the head ectoderm (hec), body
ectoderm (bec) and in the mesenchyme cells (mc).
nc
m c
bec
bec mc nt
nt
fg fg
A ee B ee
• positive reaction is observed in yolk platelet cells.
yp
yp
C

17. • In the hind brain region the reaction was localized in the head
ectoderm and in the lateral lining of the neural tube. (Fig. A)
• In the posterior part of the embryo the reaction was strong on the
outer margin of the neural tube. Somite also showed positive
reaction. (Fig. B).
• No positive reaction was seen in the notochord (n).
hec
hec bec
bec
nt
nt
nc
s
nt Nt
nc
n
s
n s lpm
A s
B

18. Discussion
• During gastrulation at 18 hrs of incubation (stage-4) the embryonic cell
start moving and get arrange into three germinal layers ectoderm,
mesoderm and endoderm.
• During this movement, cells interact with each other.
• Where as during neurulation, cells get rearranged and some of the cells
under go apoptosis.
• It is known that NOS produce NO and the presence of NO before the
synaptogenesis in the retina as well as in the brain may be considered as
signal for the development and shaping of neuronal and non-neuronal cells.

19. Hence presence of the NOS in the descrete regions of the embryo during
gastrulation and neurulation might plays an important role in signalling and
shaping of the cells.
It is also known that NOS is involved in the transmission of impulse from one
neuron to another. Presence of NOS may also indicate that in the early
development of the embryo NOS is very important in neuronal cell interaction.
Further investigations are needed to know the exact role of NOS during the early
development.