Implantation of the Organizer
in the Blastocoel
Spemann and
Mangold discovered that a piece of the organizer when placed in
blastcoel of a blastula, would be shifted to the anterior,
ventral region of the embryo by the gastrulation movements of the
host, and will induced secondary embryo in this position.
Procedure :
1.Select
gastrulae that have sickle shaped
blastopores. Remove the outer
jelly membranes. Wash the embryos
in dilute medium. Transfer
embryos to an operation dish with
a higher concentration.
2. With a glass
needle, make two flat wide groves
in the agar bottom, they should
be close together, so that both
embryos are in the visual field.
The grooves should be larger in
diameter than the gastrulae, and
not too deep. Smooth edges with a
ball tip. Place donor and host in
the grooves, they will orient
themselves with the animal pole
upward because they rotate freely
in the perivitelline space.
3. Prepare the
transplant – Remove the
vitelline membrane from the donor
gastrula. With a hair loop, turn
the embryo over so that the
dorsal blatoporal lip faces you.
With a glass needle, cut out a
sizable piece of the upper lip.
4. Prepare the
host – Grab the vitelline
membrane at the animal pole with
a pair of forceps. Tear a hole in
the membrane but do not remove
it. This hole can be used for
implantation.
5. Implantation
– Lift the transplant from
the donor and turn it upside
down. With a glass needle enlarge
the slit in the roof of the host
gastrula. Lift the transplant
with the tip of the needle. Try
to gently place it into the hole
with the glass needle. Push the
transplant with the tip of the
needle of the loop on top of the
donor. Very gently work it into
the hole with the tip of the
glass needle. Push the transplant
to the ventral side of the
blastocoel in a deep position. If
the transplant remains near the
hole, it may interfere with the
healing process.
6. Once healed,
replace the concentrated solution
stepwise by dilute solution.
7. Leave the
embryos undisturbed, but check
them several times a day. After
gastrulation is completed, turn
the embryo dorsal side up to
allow neural folds to develop
normally.
8. When neurula
stages are reached, watch for
induced neural plates and folds.
