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Studies on the mechanism of teratogenic action of trypan blue.

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STUDIES ON THE MECHANISM O F TERATOGENIC
ACTION O F TRYPAN B L U E 1
JAMES G. WILSON, ALLAN R. BEAUDOIN AND H. JAMES FREE'
Department of Anatomy, College of Medicine,
University of Florida, Gainesville
T W O FIGURES
Numerous teratogenic agents are known to cause maldevelopment of the embryos in several species, but excepting
a few spec,ific antimetabolites and antagonists (Landauer,
'54; Nelson, '57; and Murphy, Dagg and Karnofsky, '57)
little is known about the mechanisms of action; that is, where,
when, and how these agents affect the embryo. Discovery of
the teratogenicity of trypan blue (Gillman, Gilbert, Gillman
and Spence, '48) provided an agent which seems to offer
several advantages for investigating mechanisms of teratogenesis: it is easily applied, it produces a high yield of defective offspring, it has numerous closely related analogues,
and it is relatively innocuous to the pregnant mother. I n the
past 10 years a considerable volume of literature on its use
has accumulated, but this has been primarily descriptive, with
scant attention to the manner of action of the dye. Two recent studies are exceptions in that they provided information
on the probable site of action. Ferm ('56) demonstrated in
the rabbit that trypan blue traversed the blastocyst wall and
was present in appreciable amounts in the yolk sac fluid on
the 7th, 8th, and 9th days of pregnancy, indicating that the
dye has direct access to the embryo during early stages of
differentiation. The dye is teratogenically active in the rabbit
at this time. Beaudoin and Wilson ( '58) found that injection
Supported by N.I.H. Grant A-1090 from the National Institute of Arthritis
and Metabolic Diseases.
* Lederle Medical Student Fellow.
115
THP. ANATOXICAL RECORD, voL.
FEBRUARY 1959
133, No. 2
116
JAMES G. WILSOK A N D OTHERS
of the dye into chicken eggs at 24 or 36 hours of incubation
resulted in high rates of malformation among survivors, proving that the dye can exert direct teratogenic action on a n
embryo, without the mediation of a maternal organism or a
placenta. This indircct evidence and the lack of indications to
the contrary allows the assumption that trypan blue acts
directly on the mammalian embryo. The following experiments represent further attempts to elucidate the mode of
teratogenic action of trypan blue.
METHODS
All experiments were carried out using the same sample
of specially piirified trypan h l ~ e .F~r e s h solutions were prepared every two weeks i n sterile distilled water or sterile
saline and kept refrigerated except just prior to use. Injection and operative procedures were done under semisterile
conditions. Rats were from two comnwreially available stocks,
Hamilton F a r m s and Rockland Farms. Pregnancy was regarded as of one day's duration 2 1 hours after the morning
on which sperni were found in the vaginal smear. Laparotorny
was usually performed on the 7th clay to verify pregnancy and
count implantation sites. Prcgriancy was routinely interrupted on the 20th day, one day prior to term, so that all surviving fetusw could be uecoac.red intact. After removal frorn
the uterus the fetuses were weighed, examined for gross malformations, and prescrvecl. Snhsequently many of the fetusw
were sectioned by making I-mm slices through the head and
trunk with a razor blade and the slices then examined under
a dissecting microscope.
RESULTS
Time of actioii of t h c dye. Gillrnari et al. ('31) concluded
that trypan blue coixlcl cause mnldevelopmeiit of the off spring
when administered to female rats a t any time during the first
9 days of pregnancy and, to a limited extent, even when given
Purificd and donated through the courtesy of Mr. Floyd Greene of Matherson,
Culrman a n d Bell Company, Norwootl, Ohio.
T E R A T O Q E N I C A C T I O N O F T R Y P A N BLUE
117
prior to conception. However, the dye was injected at 14-day
intervals, both before and during pregnancy, which precluded
reliable observations on its effectiveness at any particular
stage. I n the present study treatment was restricted in time
so that the period of greatest susceptibility of the embryo
could be determined. I n one experiment a subcutaneous injection was given on each of three consecutive days, each injection consisting of 1ml of 1%aqueous solution of trypan blue
(total dose - 30 mg d r y ~ t 7 t . ) . Groups of approximately 10
young nulliparous females were treated in this way beginning
on day 0, 2, 4, 6, 7, 8, 9, 10, 11or 12 of pregnancy.
5o
40 -
---A
3 Subcutaneous injections on
-0
Single intravenous injection
on day indicated
consecutive days beginning
on day indicated
I
I
I
/
,+?I
I
't
The results of these three-day treatments a r e presentecl
in figure 1 (broken line). Teratogenic effectiveness of the dye
increased steadily when the initial injection was given at
progressively later dates in pregnancy from day 0 to day 7 but
decreased precipitously after day 7, and by day 11no activity
could be demonstrated. These data indicate that treatment
begun on day 7 and continued on days 8 and 9 spans the most
susceptible period of the embryo but they do not reveal the
day on which maximal teratogenic action occurs.
118
JAMES G . WILSON AND OTHERS
I n order to determine the time of maximal effect single
intravenous injections of dye were given on specific days of
pregnancy. One milliliter of 2% aqueous solution (20 mg dry
wt.) was given to groups of approximately 10 pregnant rats
on days 6, 7, 8, 9, 10, 11or 12. The results (fig. 1, solid line),
although closely paralleling those obtained with subcutaneous
TABLE 1
Frequency of various malformations after subcutaneous injection with t q p a n
blue a t different times in pregnancy
DA YS OF PREC~NANCYON WHICH TREATMENT W A S GrVEN
2
3
4
4
6
5
6
7
8
2
8
9
8
9
10
9
10
11
6
5
10
12
11
16
26
37
111
49
10
18
10
9
4
4
0
0
0
0
70
10
0
0
0
10
0
0
20
0
0
75
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
25
0
0
0
0
0
0
0
0
1
Number of
malformed animals
% malformed
7
1 0 1 1 1 2
1 1 1 2 1 3
12 13 14
% of animals with following defects *
Hydrocephalus
Short tail
Short trunk
Anophthalniia
Microphthalmia
Meningocele
Exencephaly
Gastroschisis
Facial defects
Foot defects
Spina bifida
54
66
40
9
0
30
18
0 40
27
17 20
17
10
0
0 0 1 8
0
10
0
0 0 1 8
0
0 3 6
0 2 0
0
0
20
0
42
8
19
13
27
4
12
0
0
8
8
55 50
21 30
13 10
13 30
6
0
7 3 0
6
20
5 1 0
3
0
5
0
3' 10
0
0
0
'Other types of malformations such a s those affecting the heart and major
arteries also occurred but are omitted from the table because they were less
obvious and not routinely sought f o r in all groups.
injections, clearly indicate that the peak of teratogenic activity
falls on the 8th day. The disparity in height of the curves
probably reflects the difference in total dose of the dye administered, there being close correlation between the ratio
of dry weight of dye (30 mg: 20 mg) and the maximal teratogenic activity (49 % : 32% ) .
Stability of the pattern of nzalformatiam a f t e r treatmen,t
a t different times. Table 1presents the frequency with which
TERATOGENIC ACTION O F TRYPAN BLUE
119
various types of malformations occurred when subcutaneous
injections were given in three-day sequences at different times
in pregnancy. The over-all percentage of abnormal young
varied considerably with the time of treatment, as shown in
figure 1,but the relative frequencies of individual defects did
not consistently increase or decrease as pregnancy advanced
from day 0 through day 10. F o r example, hydrocephalus was
always the most commonly occurring defect and gastroschisis
one of the rarest, regardless of when treatment was given.
Thus, it appears that trypan blue causes approximately the
same types of malformations, whether given early or late in
the period during which a teratogenic effect can be elicited.
This constancy in over-all pattern of abnormalities suggests
that the dye acts only within a limited span of time. If this
were not the case, the pattern would be expected to change as
different presumptive regions of the embryo underwent critical
phases of differentiation and organogenesis. Such changes in
pattern have been observed following maternal vitamin A
deficiency (T'Vilson, Roth and Warkany, ' 5 3 ) , use of folic acid
antagonist (Nelson, Wright, hsling and Evans, ' 5 5 ) ,x-irradiation (Russell, '50 and Wilson, '51) and other agents known to
act over a period of several days.
The composition of the pattern of malformations produced
by trypan blue provides a clue as to when the postulated
limited span of action might occur. It has been emphasized
(M7ilson, '55 and Fox and GOSS,'56) that a preponderancp of
the defects occur in the central nervous system, including tlie
eye, the cardiovascular system, and the caudal part of the
vertebral column. Primordia of these structures are among
the first to differentiate morphologically on the 9th day of
gestation and it is very likely that they are t o some degree
chemically differentiated during the 8th day. Since dye administered on the 9th day and subsequently has little or no
teratogenic effect, it may be concluded that the dye acts primarily during the period of earliest organ differentiation,
that is, late in the 8th and perhaps early in the 9th days.
120
JAMES G. WILSON A N D OTHERS
Direct exposure of the enzbrpo t o t r p p a n blue. To detcrmine whether embryos younger than 8 days a r e capable of
reacting to the dye when directly exposed in utero, solutions
of dye mere injected into the uterus of rirtts o n the 5th and
6th days of gestation. Since implantation occurs during the
6th clay, injection at or prior to this time would bring the dye
solntion into contact with the blastocysts as they float freely
in the uterine cavity or begin to settle into implantation crypts.
Both uterine horns werc ligated at their cervical ends and
then injectcd with a 27 gauge hypodermic needle at the tuba1
ends, after which another ligature as placed just below the
site of injection to prevent leakage. I n each female a small
volume of trypan blue solution (0.025 or 0.05 ml) was injected
into one horn and an equal volume of sterile saline into the
other as control. Implantation sites were not visible at the
time of injection, and the nuniber of embryos was assumed
to be the same as the nnmher of metrial glands found on the
20th day. I n 4 animals both ends of both uterine horns were
ligated but no injections were made. The ligatures alone had
no adverse effect on pregnancy or the offspring.
When injection was done on the 5th day, very few offspring
survived until term after either trypan bluc or saline injection,
regardless of volume used (table 2). Since the results with
trypan blue and saline were the same, it is concluded that
the injection and not the nature of the solution mas responsible
for preventing further development. 112 utero injections with
a 0.1% solution on the 6th day gave similar findings i n that
trypan blue caused no greater effect than a n equal quantity
of saline. When the concentration of the dye was raised to
0.5%, however, mortality among dye-treated embryos was
approximately twice that of saline controls. Therefore, the
dye in sufficient eoncentration is able to affect the 6-day embryo
by direct intrauterine exposure, but the effect was one of
increased mortality and not maldevclopment, thus tending to
substantiate the above intimation that embryos younger than
8 days a r e refractory to the teratogenic action of trypan blue.
121
TERATOGENIC ACTION O F TRYPAN BLUE
P e r s i s t e n c e of d y e in s e r u m following i n j e c t i o n . I f trypan
blue does act only during a limited period on the 8th and perhaps the early 9th days, the teratogenicity of dye injected at
earlier times would probably depend on the persistence for
several days of appreciable quantities in the maternal serum.
To determine the extent to which this actually could occur,
non-pregnant adult females were divided into three groups
each of which was injected by a different route, either subTABLE 2
I n utero injections prior t o and during implantation
Trypan hlue solution was injected into one and sterile saline into the other uterine
horn of the same female
UTZRINE H O R N S INJECTED W I T H
DAY
5th
6th
DOSE
Trypan blue
No. oF
LITTERS
Total
%
embryos Refiorbed
’
Sterile saline
56
Total
%
embryos Resorbed
%
*‘E
Z-
0.02 ml
0.1% sol.
8
41
93
0
42
95
0
0.05 ml
0.1.% sol.
10
49
96
2
56
100
0
0.05 ml
0.1% sol.
10
52
38
0
51
43
0
0.05 ml
0.5% sol.
10
52
89
0
51
47
0
I n 4 pregnant rats the uterine horns were ligated b u t no injections made. OP
41 implanted embryos in these horns, 10% resorbed and none of the survivors
were malformed.
cutaneously, intravenously or intraperitoneally. The dosage
was standardized at 1mg per 15 gm of rat regardless of how
injected, but the subcutaneous dose was divided and given in
two injections 24 hours apart to more closely approximate
treatment by this route in the preceding experiments. The
rats were killed from 1 to 72 hours after injection and the
concentration of dye in the serum determined colorimetrically.
Both intravenous and intrapcritoneal injection resulted in
very high initial serum levels which fell rapidly but remained
122
JAMES G. WILSON A N D OTHERS
above 250 p g per ml during the first 24 hours (fig. 2). Subcutaneous injection also caused a sharp rise during the first
hour but the concentration did not reach levels comparable to
those obtained by the other routes until after the second injection. The most significant conclusion to be drawn from these
data, however, is that the serum concentration of dye remains
moderately high for several days after injection, regardless of
-
0 Intravenous
-.-
u
--- A
4
8 12 16 20 24 30 36
Fig. 2
lntraperitoneol
Subcutaneous
40
Hours after ii-yecfion
60
72
Serum levels of dye a f t e r different injection routes.
how administered. The slowed rate of fall observed between
48 and 72 hours evidently continues for an extended period
of time since measurable quantities of dye have been found
in the serum 4 to 5 months after treatment with a teratogenic
dose. The means by which the dye is lost from the serum has
not been studied, although it is well known that appreciable
quantities accumulate in the reticuloendo thelial system soon
after injection. It is assumed that this is slowly liberated
back into the body fluids over the succeeding months, in which
TERATOGENIC ACTION O F TRYPAN BLUE
123
case one might expect a persisting but diminishing level of
teratogenic activity for days or even weeks after the injection
of a large dose. Indeed, Gillman, et al., appear to have
demonstrated this in reporting that a few defective young
were born to females treated before conception only. I n this
light, the steadily rising teratogenic effectiveness of trypan
blue observed to occur between conception and the 7th day
(fig. 1) can be explained in terms of higher maternal serum
levels as the time of treatment approaches the 8th day. I n
other words, the earlier the injections are given the lower
will be the concentration of dye in the maternal blood when
the embryos become susceptible on the 8th day, with a corresponding reduction in teratogenic effect.
Rete&o+t of the dye by the yolk-sac epithelium. Everett
( ' 3 5 ) noted that trypan blue injected intravenously into rats
on days 11 to 16 of pregnancy was never visible in the embryos, either in dissected fresh specimens or histologically
sectioned material. Accumulations of the dye were found in
the visceral epithelium of the yolk sac, however, within 20
minutes after injection. It was noted that prior to the completion of the chorioallantoic placenta on the 11th day, the
blood sinuses in the central zone (trophoblast) offer the only
route by which substances in the maternal blood could diffuse
into the yolk sac cavity and thus gain access to the embryo.
To extend these observations to earlier stages pregnant
females were injected with trypan blue on day 7 and the uteri
removed one, two, and three days later and prepared f o r
histologic study. Owing to the small size of rat embryos at
these stages gross observations on tissues and membranes o r
spectrophotometric analysis of yolk sac fluid was not feasible.
Microscopic examination revealed that by the 8th day conspicuous quantities of dye had already accumulated in the
visceral layer of the yolk-sac epithelium that lay beyond the
region of the embryo proper. Curiously, the eiidoderm immediately associated with the embryonic mass was entirely
devoid of visible dye. No dye was seen in any other embryonic
tissue or membrane. On the 9th and 10th days the distribution
124
J A M E S G. WILSON A N D OTHERS
of dye differed only in that the quantity within the extraembryonic part of visceral endoderm was noticeably increased.
By the 10th day this inverted yolk sac virtually encloses the
embryo proper, and by the 11th day the embryo is totally surrounded. The visceral yolli epithelium, thus, begins to take
up trypan blue on the 8th day of gestation and apparently
continues t o d o so until the 16th day. Since trypan blue has
been observed to persist in the yolk sac epithelium in large
amounts until term, it is possible that dye once accum:ilateJ
wit hiri the endodermal cells becomes immobilized. This being
the case the epithelium could serve to protect the embryo by
absorbing all or most of the available dye within the yolk sac
TABLE 3
Effect of in,jecfin,g particiilatc a?id 7kiglily colloidal substances i i t f o f e m a l e r a t s
o n t h e 7 t h , S t k , aid. 9t72 days of pregnancy
SUBSTAVCE
India iiik
h h g n r a blue 2B
NUNIIPR
XUMIIFR
IBJECTED
FEDIALRS
CONTINUING
T O TRRlll
10
5
10
11
O F B S P X I Y G CARRIED TO TCRhI
_-
%
Dead
58
4
116
7
%
Abnormal
0
0
lIiijccted as three l - c m 3 doses in 50% aqueous solution.
’Injected as three l - c m 3 doses in 1% aqueous solution.
fluid a€ter the 9th day of gestation, and especially after the
10th day when the embryo becomes almost completely enclosed by yolk endoderm. I t is possible, then, that the dye
has access to the embryo only before the yolk endoderm begins
to accumulate it in appreciable quantity, or at the latest, before this layer encloses the embryo.
Possible “blockage” of matermd reticulo-e~~zdothelialsystem. It is well established that trypan blue is avidly taken
up by phagocytes of the rcticuloendothelial system. Since
several non-teratogenic azo dyes were shown not t o be phagocytized (TTilson, ’55), the possibility exists that the tcratogenic activity of trypan blue might somehow he related t o a
saturation or “blockage ’’ of the maternal reticuloendothelial
system. To test this possibility, Niagara blue 2R, a non-
TERATOGENIC ACTION O F TRYPAN BLUE
125
teratogenic dye with chemical structure and colloidal properties similar to trypan blue, arid India ink, a particulate
suspension, were injected into pregnant rats on days 7, 8,
and 9 of gestation. These substances did not cause malformations (table 3 ) , although India ink resulted in a high r a t e of
maternal death. Since both substances mere taken up by the
maternal reticidoendothelial system in great quantities, it may
be concluded, therefore, that loading the phagocytes of this
system with colloidal or particulate substances has nothing
to do with teratogenesis.
DISCUSSIOX
The evitlence at hand suggests that trypan blue exerts its
teratogenic action directly on the rat embryo and that this
action o c c ~ ~for
r s only a limited time during the period of early
differentiation. Snpport was lent to the idea of direct action
on the embryo by F e r m ('56) who showed that the dye can
traverse the blastocyst wall of the 7- to %day rabbit embryo
and accumulate in apprccialnle quantity in the yolk sac fluid.
Additional support comes from the observation by Eeaudoin
a n d Wilson ( '58) that the developing chick became malformed
when the dye was injected in proximity to the embryo (luring
carlp stages of incubation.
The contention that trypan hlue exerts its teratogenic effect
during a limited span in gestation is hascd in p a r t on circumstantial cvidence, and i n p a r t on direct observation. The fact
that the loss of tpratogenic activity after the 8th day is correlated in time with the active absorption and accumulation
of the dye by the visceral layer of yolk sac endoclerm suggests
that this structure is somehow involved. Absorption of the
dye by the endoderm has been observed to occur from the
8th through at least the 16th clay. This layer virtually surrounds the embryo from the 10th day onward. If it is assumed
that the epithelial cells retain the absorbed dye, a mechanism
would exist for preventing the dye from reaching the emlnrj-o.
Such an hypothesis implies the further assumption that the
chorioallantoic placenta fails to transmit the dye after that
126
JAMES G. WILSON AND OTHERS
structure becomes functional. These possibilities are currently being tested by using radioactive trypan blue and autoradiographic techniques .
That the dye has its greatest effect on the rat embryo during
the 8th day of gestation has been amply demonstrated. Treatment at this time has maximal teratogenic effect, coincides
with beginning organ differentiation, and affects almost exclusively those organ systems first differentiated, namely, the
central nervous system, the heart and major arteries, an3
the axial skeleton.
Despite the occurrence of malformations following treatment given prior to the 8th day, an immediate teratogenic
effect on the embryos at these earlier times has been questioned. I n the first place, attempts to produce developmental
defects by exposing 6-day embryos t o the dye directly by injecting it irz utero were unsuccessful, although the dye did have
a demonstrable effect on mortality. Secondly, an effect prior t o
the 8th day would be inconsistent with the beginning of teratogenicity observed f o r other agents used in the rat. X-irradiation (Wilson, '53) maternal vitamin A deficiency (TT7ilson,
Warkany, and Roth, '53) and maternal folic acid deficiency
(Nelson, Wright, Asling and Evans, '55) all cause malformations in rat offspring only when treatment was given on the
9th clay or later. Undoubtedly the failure of these agents to
cause abnormal development mas related to the fact that the
rat embryo does not begin organ differentiation until late in
the 8th or early on the 9th day. At earlier ages teratogenic
agents probably either affect the undifferentiated cells in such
numbers that the embryo dies, or if relatively few cells are
damaged, repair is possible and the embryo develops normally.
I n order to produce discrete malformations in mammals it,
Tvonld appear that teratogenic agents must act on cells with a
prcdetermined role in development, in other words, induction
or chemical differentiation must have occurred before the
primordial cells are rendered abnormal. Thus, trypan blue
would be unique among known teratogens if it were effective
before differentiation began.
T E R A T O G E N I C A C T I O N O F TRYPAN BLUE
127
SUMMARY
Trypan blue has its maximal teratogenic effect in the rat
when administered on the 8th day of gestation, but some effect
followed treatment at all times from the day of conception
through the 10th day. The percentage of defective offspring
rose steadily after injection on successive days up to the 8th
day, after which it fell rapidly until the 11th day when malformation could no longer be produced.
Available data indicate that the dye does not act on the
embryo prior to the 8th day. The effects produced by earlier
treatment can be attributed to residual dye in the maternal
serum since dye was demonstrated to remain at moderately
high serum levels for several days after the injection of a
teratogenic dose. Direct exposure of implanting embryos to
dye injected in utero on the 6th day failed to cause malformations, although it was possible to increase the mortality rate
by such treatment. The pattern of malformations was shown
t o remain constant regardless of when treatment was given,
strongly suggesting that the dye acts on the embryo during
a limited span of time.
The abrupt cessation of activity after the 8th day, at a
time when other teratogenic agents are most active, appears
to be related to the development of the visceral layer of extraembryonic endoderm, the so-called yolk-sac placenta. This
structure progressively encloses the embryo and at the same
time absorbs and stores large quantities of the dye. It is postulated that the dye is immobilized in the yolk endoderm,
thereby protecting the embryo from further teratogenic influence.
Loading or “blockage ? ’ of the maternal reticulo-endothelial
system with India ink and other colloidal but non-teratogenic
dyes was shown not to be concerned with teratogenic activity.
LITERATURE CITED
BEAUDOIN,
A. R., AND J. G. WILSON 1958 Teratogenic effect of trypan blue
on the developing chick. Proc. SOC.Exp. Biol. Med., 97: 85-90.
EVERETT,
J. W. 1935 Morphological and physiological studies on the placenta
in the albino rat. J. Exp. Zoo]., 7 0 : 243-285.
128
J A M E S G. WILSON A X D OTHERS
FERN,
V. 13. 1956 Permeability of the rabbit blastocyst to trjpaii blue. Anat.
Rec., 126: 745-760.
AND C. M.Goss 1936 Expezirneiital production of a qndrome of
congenital cardiovascular defects in rats. Ibid., 1 2 l : 189-208.
GILLXAN,J., C. GILBERT,T. GILLMANAND I. SPENCE1948 A preliminary report
o n hydrocephalus, spina bifida aiid other congenital anoinalies in the
rat produced by trypan blue. S. Afr. J. Mcd. Sci., 13: 47-90.
GILLMAN,J., C. GILBERT, 1. SPENCE
AND T. GILLMAN 1951 A further report on
anomalies in the r a t produced by trypan blue. Ibid., 1 6 : 125-135.
LAND~ U E R ,W. 1954 On tlie chemical procluctioii of clevclopinental abnorrnalities
aiid of plicnocopies i n cliickeii embryos. J. Cell arid Conip. Phpiol.,
43: 261-305, (suppl. I).
MUXPIIY,&
L.,
I.
C. P. DAGGAND D. A. KARNOFSKY
1957 Comparison of teratogeitic chemicals in the rat aiid chick embryos. Pediat., 1 0 : 701-714.
XELSON, M. M. 1957 Production of congenital anonlalies in iiiarnmals by maternnl dietary tleficiencies. Ibid., 19 : 764-776.
X E L ~ O N124.
, $I
IT..W.
, WRIGIIT, C. W. ASLINGAND 1%M. EVANS1955 ?Ilultiplc
congenital ai~liormaiitiesresulting from transitory deficiency of ptcroylglutainic acid during gestation in tlie rat. J. Nutrition, 56: 319-370.
RUSSELL,L. I3. 1950 X-ray induced abnormalities i n tlie mouse and their use i r i
the analysis of einbryologieal patterns. I. External and gross visceral
changes. J. Exp. Zool., 114: 515-602.
WILSON,J.G . 1954 1):fierentiation aiitl the reaction of rat enibrgos to radiatroit.
J. Cvll. and Conip. PI13 siol., 43: 11-38, (suppl. 1 ) .
1935 Teratogcnic. actiLity of several a m dyes clieinically relntetl
to tlgpaii L l ~ t , . A1i:lt. R ~ c . ,123: 313-334.
WILSON,J . G., C. B. ROTH A K D J. W A R K A N Y 1953 An analysis of the syntlroriie
of nialforniations induced h j niatcinal vitaniiii 9deficiency. Effects of
restoration of vitaniin h a t various times during gestation. Am. J.
Anat., ‘ I ? : 159-218.
Fox, hl. II.,
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