/ . Embryol. exp. Morph., Vol. 17,1, pp. 161-169, February 1967
Printed in Great Britain
Studies on induction
by substances containing sulphydryl groups in
post-nodal pieces of chick blastoderm
By M. ABDUL WAHEED & LEELA MULHERKAR 1
From the Department of Zoology, University ofPoona
The importance of sulphydryls in morphogenesis is being increasingly recognized. In the study of the role of thiols in amphibian and avian development one
of the methods of approach has been to subject early embryonic stages to the
action of reagents such as chloroacetophenone, monoiodoacetic acid, oxidized
glutathione and mercaptoethanol (Beatty, 1949; Rapkine & Brachet, 1951;
Deuchar, 1957; Brachet & Delange-Cornil, 1959; Pohl & Brachet, 1962;
Lakshmi, 1962a). One common effect seen in most of the above experiments is
that in treated embryos the nervous system is affected, in that it remains an
open thick plate.
Using chloroacetophenone, a specific SH inhibitor, on chick embryos Lakshmi
(19626) has shown that it causes a marked decrease in the inducing capacity of
Hensen's node. Chromatographic analysis of chloroacetophenone-treated primitive streaks showed that cysteine and glutathione were affected most (Lakshmi &
Mulherkar, 1963). Moreover the effect on chick morphogenesis was found to be
reversible by subsequent treatment with cysteine hydrochloride (Mulherkar,
Rao & Sanjivani, 1965). It was thus demonstrated that chloroacetophenone
inhibits neural induction by the blocking of SH groups in the organizer while by
the addition of an SH-containing substance such as cysteine hydrochloride the
block is reversed.
Earlier Brachet & Rapkine (cited by Brachet, 1950) increased the concentration of SH groups in ventral explants of amphibian gastrulae by treating them
with cysteine, with glutathione in its reduced state, or with thiomalic acid, and
obtained the formation of a nervous system, but Brachet (1950) expressed doubt
as to the use of cysteine in such experiments in view of its toxic effect in the seaurchin egg. In the present experiments SH-containing substances such as cysteine
hydrochloride and glutathione in the reduced state are used in a chick embryo
system at a concentration which is less toxic. Brief periods of treatment at low
concentrations showed no visible toxic effect on morphogenesis. The post-nodal
1
Authors' address: The Department of Zoology, The University of Poona, Poona, 7, India.
II
JEEM 17
162
M. A. WAHEED & L. MULHERKAR
pieces 0-8 mm behind the node which do not normally possess inductive
capacity were tested to see whether they acquire inductive capacity as a result
of the chemical treatment.
MATERIALS AND METHODS
Fresh, fertilized White Leghorn eggs were incubated at between 37-5 and
38 ° C. The definitive primitive streak stage was used when at a length of
between 1-9 and 2-1 mm. All instruments and containers used in the culturing
and in the operations on embryos were sterilized. The component solutions
used in the preparation of Pannett Compton's saline (PC saline) were autoclaved
in separate containers.
Short treatments (about 6h) with 001 % L-cysteine hydrochloride and
0-025 % reduced glutathione were found quite harmless and at the same time
effective.
The embryos were explanted, by the procedure described by New (1955),
when the length of the primitive streak was about 1-8 mm (0-3 mm short of full
streak length). They were treated either with cysteine hydrochloride (0-01 %) or
glutathione (0-025 %) and incubated for a further 6 h to attain the definitive
streak stage. This duration of incubation is also the period of chemical treatment. Equal numbers of untreated embryos served as controls. When the
primitive streak had fully formed the solution of cysteine or glutathione was
pipetted out, two or three changes with PC saline were given and a few drops of
fresh PC saline were added. The embryos were allowed to grow for a further
18 h. Such brief treatments with cysteine or glutathione at the concentrations
used did not cause any toxic effect on the morphogenesis of the treated embryos
as compared with the controls.
Grafting experiments
The host embryos employed for grafting were incubated until they attained
definitive primitive streak stages. The donor embryos were treated with the
desired strength of either cysteine hydrochloride (001 %) or glutathione
(0-025 %) as described earlier. The blastoderms were detached from the vitelline
membrane and rinsed three times in fresh PC saline. The length of the primitive
streak was measured with a micrometer. A square graft of 0-2 mm was cut out
at a level 0-8 mm behind the reference line (see Fig. 1). The inductive capacity
of such post-nodal grafts was tested by grafting each of them into a host embryo
by the method of Waddington (1932). Similar untreated post-nodal grafts
served as controls. In all, 81 experimental grafts (41 treated with cysteine and
40 with glutathione) were made and 63 controls (about 30 for each chemical)
were maintained.
The grafted embryos were incubated for 18-20 h, after which they were fixed
in Bouin's fluid, and serially sectioned at 10 fi for histological examination.
Induction by sulphydryls
163
EXPERIMENTAL RESULTS AND DISCUSSION
There were two points under investigation in these experiments: the effect of
SH-containing substances on (a) the capacity for neural differentiation of the
post-nodal graft itself, and (b) the inducing capacity of the treated grafts.
Donor (treated)
Host (untreated)
j
10
i
5
Fig. 1. Diagram to show the scheme of grafting.
07 mm.
,
^a^U._
" GNT
"•'4""
Fig. 2. Transverse section of embryo. Cysteine-treated post-nodal graft showing
induced neural plate (INP) and neuralization of graft tissue (GNT). HN, host neural
tube.
HN
.
P
0-2 mm.
GS
Fig. 3. Transverse section of embryo. Cysteine-treated post-nodal graft showing
palisade induction (P) and somite formation (GS) in graft tissue. HN, host neural
tube.
164
M. A. WAHEED & L. MULHERKAR
Cysteine hydrochloride-treated grafts
Fifteen out of forty-one (32 %) of the cysteine-treated post-nodal grafts
showed neuralization of their tissue (Fig. 2): twenty-three grafts caused neural
induction in the host ectoderm (Figs. 2-5). The majority of the thirty control
grafts were absorbed in the host tissue. A few of them (about 10 %) kept their
identity and were seen in the form of mesodermal cells. Two control grafts
induced palisade-like formations in the host ectoderm.
INP
Ji^ :l f v
Fig. 4. Transverse section of embryo showing induced neural plate (INP) and
host neural tube (HN)."
:•;„--•—
..?®m>- •*::.
Fig. 5. Transverse section of embryo. Cysteine-treated graft showing induction
(INP) and somite formation of graft tissue (GS). HN, Host neural plate.
^HN
^mWttlt:, X" '-.407% \
•/' ^I^**^ *•
G
V.»
^;;«^f
Fig. 6. Transverse section of embryo. Glutathione-treated post-nodal graft (G)
causing induction (INP) in the host ectoderm. HN, Host neural tube.
Reduced glutathione-treated grafts
Seventeen per cent of the treated grafts showed neural differentiation of their
own tissue; 57 % of the grafts caused inductions in the host ectoderm (Figs. 6,7).
Induction by sulphydryls
165
As in the case of cysteine controls, the majority of the thirty-three controls in
this series were absorbed into the host tissue. Three control grafts, which themselves formed mesoderm, induced palisade-like structures in the host ectoderm
(Fig. 8). The results of both series are given in Table 1.
0-2 mm.
Fig. 7. Transverse section of embryo. Glutathione-treated post-nodal graft (G)
causing induction (INP) in the host ectoderm. HN, Host neural tube.
Fig. 8. Transverse section of embryo. Control post-nodal graft (G) showing palisade
formation (P) in the host ectoderm. HN, Host neural plate.
Table 1.
Treatment
Total no.
of grafts
made
Induction
in
Neuralized Mesodermal
Absorbed
Cysteine
Experimental
Control
41
30
23
2
(Palisadelike)
15
None
15
8
11
22
Glutathione
Experimental
Control
40
33
25
3
(Palisadelike)
7
None
25
7
8
26
DISCUSSION
The capacity for the formation of neural tissue in the region behind the node of
definitive streak-stage chick embryos has been studied by a number of investigators using various methods. Using the chorioallantoic technique Hunt (1931)
166
M. A. WAHEED & L. MULHERKAR
and Rawles (1936) fixed the limit of neural differentiation 0-5 and 0-3 mm
behind the node. Waddington (1935) and Mulherkar (1958) obtained neural
tissue in vitro as far back as 0-7 mm behind the pit. However Rudnick (1938)
and Spratt (1952) found that neural plate never developed in posterior pieces
about 0-4 mm behind the pit.
On the basis of these findings post-nodal pieces taken from a level 0-8 mm
behind the node region, which in normal development are destined to form
lateral plate mesoderm rather than neural tissue, were employed. Inductive
capacity has been shown to be associated with the presumptive axial mesoderm
and also with the capacity of the graft to form neural tissue. Therefore it is
reasonable to suppose that grafts taken 0-8 mm behind the node will not normally
differentiate into neural tissue or cause neural inductions unless such a capacity
is newly acquired by them after treatment with cysteine or glutathione, although
slight inducing capacity was reported by Abercrombie (1954) and Ahsan (1956)
from the non-inducing two-thirds portion of the definitive primitive streak. In
the present experiments it has been found that a substantial number of postnodal grafts treated with cysteine or glutathione have differentiated into neural
tissue and somites (Figs. 4, 5).
The observations regarding the effect of sulphydryl compounds, and particularly of cysteine hydrochloride, in promoting somite formation are in agreement with those of Fraser (1960).
It could be that cysteine and glutathione exert their neuralizing effects because
of their toxicity as has been shown at higher concentration and longer duration
of treatment. The present concentration of 0-01 % cysteine hydrochloride is
much lower than that used by Brachet & Rapkine (1941), who used 0-1 M. Also,
the duration of treatment given in the present experiments is very short. Moreover, thirty whole embryos subjected to treatment with the two chemicals as
described earlier showed more or less normal development.
The percentage of inductions by post-nodal pieces treated with cysteine and
glutathione is quite high. The post-nodal grafts in the control series are seen to
be incorporated in the mesoderm of the hosts and only a weak palisade type of
induction is seen in a few cases in the controls kept for both the chemicals. It is
therefore reasonable to suppose that the inducing capacity shown by the postnodal pieces treated with cysteine and glutathione is due to their treatment with
SH-containing substances.
Both cysteine and glutathione contain amino groups as well as SH groups.
It will be interesting to see whether the observed effect is due to SH groups or
amino groups in the compounds. Some preliminary experiments in this direction
using thiomalic acid (Muzumdar, 1966; Mulherkar, Joshi & Diwan, 1966), which
contains only SH groups, suggest that it is SH groups rather than amino groups
that are important in the morphogenesis of nervous system. Research work
along this line is in progress in this laboratory.
Induction by sulphydryls
167
SUMMARY
1. Chick embryos at mid-primitive streak stage were explanted in vitro by
the method of New and treated with L-cysteine hydrochloride (0-01 %) or
reduced glutathione (0-025 %). Each substance was kept in contact with the
embryo for about 6 h, after which the embryos showed normal development
when cultured in Pannett Compton saline.
2. Post-nodal primitive-streak pieces taken from a level 0-8 mm behind the
node region of the treated embryos were grafted into normal host primitivestreak chick blastoderms according to the method described by Waddington.
Control grafts of untreated pieces were made.
3. For each series about 75 grafts were made of which 40 were experimental
and 35 were control. Experimental grafts treated with cysteine hydrochloride
produced inductions in 56 % of cases. Grafts treated with glutathione caused
inductions in about 63 % of cases. Control grafts in both cases produced at most
palisade formation in the host ectoderm, and this only in negligible proportions:
32 % of the cysteine-treated grafts and 17 % of glutathione-treated grafts underwent neuralization. While most of the control grafts were absorbed into the
host mesoderm, a few gave rise to somites.
4. It is suggested that the inducing capacity acquired by the post-nodal
pieces is due to their treatment with substances containing sulphydryl groups.
Factors such as toxicity of the chemicals and the effect of host individuation field
are ruled out.
RESUME
Les effets inducteurs de substances -SH dans des fragments
post-nodaux a" embryons de poulet
1. Des embryons de poulet au stade de la ligne primitive intermediate ont ete
explantes in vitro selon la methode de New et traites par l'hydrochlorure de
L-cysteine (0-01 %) ou par le glutathion reduit (0-025 %). Apres un traitement
de 6 h, les embryons se developpaient normalement.
2. Des fragments post-nodaux, decoupes environ 8 mm derriere la region
nodale d'embryons prealablement traites, furent greffes selon la methode decrite
par Waddington, sur des embryons notes au stade de la ligne primitive. Des
fragments non traites et greffes de la meme maniere ont servi de temoins.
3. Chaque serie de 75 greffes comportait 40 traites et 35 temoins. Dans le
cas des temoins, l'ectoblaste de l'hote n'a forme que des cellules palissadiques,
et cela en nombre reduit, tandis que chez les embryons traites une induction fut
observee dans 56 % des notes ayant une greffe pretraitee par l'hydrochlorure
de cysteine, et dans 63 % des cas pretraites par le glutathion. Parmi les resultats
positifs, nous avons observe une neuralisation dans 32 % des cas en ce qui
concerne la cysteine, et dans 17 % des cas en ce qui concerne le glutathion. Les
168
M. A. WAHEED & L. MULHERKAR
greffes temoins etaient, dans la plupart des cas, resorbees par le mesoderme
de l'hote; dies ne produisirent que tres rarement des somites.
4. II a ete suggere que le pretraitement par des substances -SH confere une
capacite inductrice aux fragments post-nodaux. II est exclu que ces resultats
positifs soient dus a la toxicite des substances chimiques aux champs de differentiation de l'hote.
We thank Dr P. N. Joshi of the University Department of Biochemistry for valuable
discussion. One of us (M.A.W.) is grateful to the University of Mysore for the financial
support received during the period of this investigation.
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(Manuscript received 16 March 1966, revised 18 July 1966)