Dimensions of the Notochord and Somites in
Embryos of Xenopus laevis Treated with Thiocyanate
by SILVIO RANZI and
GIANNA GAVAROSI1
From the Department of Zoology, University of Milano
A s a result of previous research, Ranzi & Tamini (1939) pointed out that the
notochord of Amphibian embryos of several species treated with sodium thiocyanate (NaSCN) solutions is larger than the notochord of control embryos.
Later, several substances were identified which cause enlargement of the notochord: full data and a complete review of the literature on this subject are given
by Ranzi & Citterio (1954) and Ranzi (1957). A careful statistical study on the
number of nuclei in Rana esculenta embryos was carried out by Corti (1950).
In embryos which had been treated for 24 hours with 0-05 M NaSCN, from the
late blastula stage onwards, she found 1233 + 28 nuclei at the tail-bud stage,
whereas the controls reared in spring-water had only 1107 + 24 nuclei: these
two sets of data are significantly different (standard error of the difference
±37).
Enlargement and induction of notochord by NaSCN treatment have been
reported by Badfnez, Carrasco, & Manriquez (1954). Notochord can be obtained from ventral explants and from explants of presumptive mesoderm by
means of urea, which acts like NaSCN (Leone, 1952), or by means of NaSCN
(Ogi, 1957a). However, Ogi (1957W counted the nuclei of a few embryos of Bufo
vulgar is and JR. nigromaculata treated with NaSCN and Nal and was unable
to demonstrate an increase.
For these reasons we thought it useful to repeat the work of Corti, but on
another species, Xenopus laevis (Daudin), which appeared to be very sensitive
to substances causing alterations of development (for the effect of LiCl see Backstrom, 1954).
METHODS
Eggs laid naturally by a couple of X. laevis were used; about 93 per cent, of
the eggs were fertilized and all the controls developed normally. The eggs of
this species appear to be particularly sensitive to NaSCN; a 0 01M solution produced the familiar malformations (see Ranzi, Tamini, & Storari Offer, 1946)
and yet allowed the embryos to survive.
1
Authors' address: Istituto di Zoologia, Via Celoria 10, Milano, Italy.
[J. Embryol. exp. Morph. Vol. 7, Part 2, pp. 117-121, June 1959]
5584.7
i
S. RANZI AND G. GAVAROSI—EFFECTS OF
118
A I M NaSCN solution was made up and diluted 1 to 100 with spring-water;
Xenopus embryos at stage 8 /9 (late blastula) of Nieuwkoop (1956) were placed
in the solution at 15° C. and allowed to remain there for 2 hours, i.e. during the
first part of gastrulation. Then they were carefully washed with spring-water and
allowed to develop in spring-water.
The controls were reared in identical finger bowls in spring-water.
When the control and experimental embryos at the age of 48 hours reached the
tail-bud stage, 50 controls and 50 experimental embryos were fixed. During
development we noticed a slight retardation in the controls compared with the
NaSCN-treated embryos.
The embryos were fixed in Bouin's fluid, paraffin embedded, and sectioned at
10//; sections were stained with Ehrlich haematoxylin and eosin. The counting
of nuclei was performed with a micrometer disk mounted in a x 7 ocular and
a x 62 objective. All the nuclei which appeared in one section were counted;
the error in counting twice those nuclei which traversed two sections was then
corrected.
RESULTS
We first measured the volume of notochord in the controls and the NaSCNtreated embryos. The volumes expressed in /x3 x 106 (mean and standard error)
were 11-5 + 0-59 and 10-0 + 0-52 respectively. The difference is not significant.
20-
Control
Na SCN
15-
§
10-
5-
n
•I
701
801
901
1001
1101
1201
1301
1401
1501
800
900
1000
1100
1200
1300
1400
1500
1600
TEXT-FIG. 1. Diagram of the crude counts of nuclei in the notochord of 50 controls and
of 50 embryos treated with sodium thiocyanate (NaSCN).
In comparing the numbers of nuclei we have avoided any error due to an
accelerated development induced by NaSCN for the controls used were, in fact,
a little more advanced than the treated embryos, and many of them showed the
beginning of vacuolization in notochordal cells.
THIOCYANATE ON EMBRYONIC DEVELOPMENT
119
The results of the nuclear counts are given in Text-fig. 1. The mean number of
nuclei of the 50 individuals in each group is: control 1056-6 + 18-5; NaSCNtreated 1137-3 ±21-5. These values must be corrected, taking into consideration
the dimension of nuclei (see Abercrombie, 1946). The diameters of the nuclei are
14-1 ±0-72 fi for the controls and 13-2 + 0-67 fx for the treated embryos. Since the
thickness of the sections was 10/x, the corrected number of nuclei is therefore as
follows: controls 438-4 + 7-67; treated embryos 490-2 + 9-25. There is here a
highly significant difference (001 > P > 0001), the increase amounting to
11-8 percent.
volume of notochord
We can therefore determine the ratio
i.e. the mean
number of nuclei
volume of a cell (cells were assumed to be uninucleate). The volume, expressed
in /A3 was: controls 26,232 + 1220, treated embryos 20,400 + 567. The higher
standard error of the controls is due to the fact that some embryos had larger
notochordal cells due to greater vacuolization.
|
[control
Na SCN
7654321-
I
1 i
8001
11000
11001
14000
14001
17000
TEXT-FIG. 2. Diagram of the crude
counts of nuclei in the somites of 10
controls and of 10 embryos treated
with sodium thiocyanate (NaSCN).
Since research carried out by Cigada (1957) showed that NaSCN stimulated
the division of Amoeba, the question arose as to whether the increase of nuclei
in the notochord was a specific phenomenon, confined to this organ, or a general
120
S. R A N Z I A N D G. G A V A R O S I — E F F E C T S OF
phenomenon detectable in all the rudiments of the embryo. Unpublished work
by Ranzi & Tamini had already shown that the nuclei of the ectoderm of
NaSCN-treated embryos do not increase in number. In the present investigation
we examined the number of nuclei in the somites, from whose presumptive
material Ranzi & Tamini (1940) had shown that notochordal cells segregate. The
results are shown in Text-fig. 2. The mean number of nuclei of the 10 individuals
in each group is: control 13,750 + 565; NaSCN-treated 10,987 + 586.
The mean diameter of the nuclei of the controls was 7-9 ±0-37/*, that of the
treated embryos 7-5 ± 0-36/x, and the thickness of the sections 10/*. The corrected number of nuclei is therefore: controls 7793-4 + 315-8 nuclei; NaSCNtreated embryos 6273-3 + 334-7 nuclei. This represents a highly significant
difference (001 > P > 0-001). The decrease of the nuclear number in the somites
of the NaSCN-treated embryos is 24 • 1 per cent.
The conclusion to be drawn is that cells of the somite rudiment are transformed into notochord.
SUMMARY
The number of nuclei of the presumptive notochord and somite cells of embryos of X. laevis at early tail-bud stage was determined in normal and NaSCNtreated embryos.
The nuclei of the presumptive notochord are more numerous in the NaSCNtreated embryos by 11-8 per cent., a significant difference.
The nuclei of the somites are less numerous in the NaSCN-treated by 24-1
per cent., a significant difference.
ACKNOWLEDGEMENT
This investigation has been supported by a grant from the Consiglio Nazionale
delle Ricerche.
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T H I O C Y A N A T E ON E M B R Y O N I C D E V E L O P M E N T
121
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•
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(Manuscript received 5: viii: 58)