/ . Embryol. exp. Morph. Vol. 69, pp. 151-167, 1982
Printed in Great Britain © Company of Biologists Limited 1982
151
Neural tube development in mutant {curly
tail) and normal mouse embryos: the timing
of posterior neuropore closure in vivo
and in vitro
By A. J. COPP1, M. J. SELLER 1 AND P. E. POLANP
From the Paediatric Research Unit, The Prince Philip Research Laboratories,
Guy's Hospital Medical School, London
SUMMARY
A dye-injection technique has been used to determine the developmental stage at which
posterior neuropore (PNP) closure occurs in normal and mutant curly tail mouse embryos.
In vivo, the majority of non-mutant embryos undergo PNP closure between 30 and 34
somites whereas approximately 50% of all mutant embryos show delayed closure, and
around 20% maintain an open PNP even at advanced stages of development. A similar
result has been found for embryos developing in vitro from the headfold stage. Later in
development, 50-60% of mutant embryos in vivo develop tail flexion defects, and 15-20%
lumbosacral myeloschisis. This supports the view that delayed PNP closure is the mftin
developmental lesion leading to the appearance of caudal neural tube defects in curly tail
mice. The neural tube is closed in the region of tail flexion defects, but it is locally overexpanded and abnormal in position. The significance of these observations is discussed in
relation to possible mechanisms of development of lumbosacral and caudal neural tube
defects. This paper constitutes the first demonstration of the development of a genetically
induced malformation in vitro.
INTRODUCTION
An understanding of the mechanisms which underlie mammalian neural
tube development requires* both descriptive and experimental studies. Neural
morphogenesis has been described on many occasions (reviewed by Copp,
1982), but the use of experimental procedures such as those employed with
amphibian and chick embryos (Waddington, 1956) has not so far been possible
due to the inaccessibility of postimplantation embryos. Recently, culture
techniques have become available which allow normal in vitro development
of postimplantation rat and mouse embryos through stages which include
neural tube formation (New, 1978). It has also been shown that addition of
a teratogen, excess vitamin A, to the culture medium can result in development
of neural tube defects (Morriss & Steele, 1974). It is now possible, therefore,
1
Authors' address: Paediatric Research Unit, The Prince Philip Research Laboratories,
Guy's Hospital Medical School, Guy's Tower, London SE1 9RT, U.K.
152
A. J. COPP, M. J. SELLER AND P. E. POLANI
to study normal and abnormal neural tube development in vitro, and to subject
these stages of embryogenesis to experimental analysis, using recently developed
microsurgical techniques (Copp, 1982).
The curly tail mouse mutant (ct) produces malformations closely resembling
human neural tube defects (Adinolfi et al. 1976; Embury, Seller, Adinolfi &
Polani, 1979). Defective embryos in ct litters exhibit one or more of the
following conditions: exencephaly, lumbosacral myeloschisis (LSM; the
developmental forerunner of lumbosacral spina bifida; Warkany, 1971) and
tail flexion defects. In his original paper on the mutant, Gruneberg (1954)
suggested from morphological observations that the tail abnormalities result
from delayed closure of the neural folds at the posterior neuropore (PNP).
If this is true, then the high frequency of tail flexion defects (40-60 % of all
offspring) makes the ct mutant a potentially useful system for experimental
analysis of mechanisms underlying neural tube closure. Before the latter can
be attempted, however, it is necessary to answer the following questions concerning the pathogenesis of tail flexion defects. First, is there objective evidence
to support the hypothesis that delayed PNP closure underlies production of
tail flexion defects? Secondly, is PNP closure actually completed in ct embryos
which develop tail flexion defects without LSM (i.e. the commonest manifestation), or are these embryos characterized by small persistent open neuropores? Third, do tail flexion defects develop in embryos which are maintained
in vitro and are therefore accessible for experimental analysis? These questions
are dealt with in the present paper.
MATERIALS AND METHODS
Source and culture of embryos
The ct mutation is maintained in a random-bred closed colony. Its inheritance
most closely fits that of a recessive gene with incomplete penetrance which is
markedly affected by genetic background (Embury et al. 1979). At birth 'ct
litters' contain two types of individuals. CT offspring have obvious tail abnormalities ranging from complete twists, sometimes in combination with
open neural tube defects, to slight bends. ST offspring have completely straight
tails and are not otherwise abnormal. Embury et al. (1979) found that the
overall incidence of malformed foetuses in litters derived from CT x CT matings
is approximately 52 %, whereas ST x ST matings produce only 37 % abnormal
foetuses. In the present study, a similar comparison was made between embryos
derived from CT x CT and ST x ST matings. A third category of embryos,
from a non-mutant albino random-bred strain, A/Strong, served as controls.
Natural matings were used throughout the experiments and the day of
finding a plug was designated day 1 of pregnancy. For purposes of estimating
postcoital ages (pc) of embryos, ovulation was assumed to occur at the midpoint of the dark period, i.e. at 01.00 h.
Neural tube development in mutant and normal mouse embryos 153
Embryos were cultured from the headfold stage (1-6 somites) according to
the method of New, Coppola & Terry (1973) and Sadler (1979), with modifications as described elsewhere (Copp, 1982).
Identification of developmental defects and
analysis of PNP closure
Embryos which had developed either in vivo or in vitro were dissected from
their extraembryonic membranes in phosphate-buffered saline at various times
between 9 d 8 hpc (day 10 gestation) and 12 d 9 hpc (day 13 gestation). Each
embryo was inspected for gross malformations, immediately tested for PNP
closure, as described below, and finally fixed in Bouin's fluid. Three developmental defects were studied in particular: curly tail flexion defects, LSM and
exencephaly. Tails were considered to exhibit flexion defects if they contained
a dorsal bend of 90° or more relative to the longitudinal axis of the developing
tail. This criterion excluded many embryos (especially among those with
35-40 somites) which had tail flexions only slightly less than 90°, probably
representing an early stage in development of a curly tail. LSM appeared as
a region of non-fusion of neural folds extending in a caudal direction from
around the level of the hindlimb buds. LSM embryos were classified as having
an open PNP. LSM was easily recognizable in embryos with a substantial
number of caudal somites (i.e. total somite number of 40 or more), but in
embryos at earlier stages of development LSM was difficult to distinguish
from (and may originate as) an extreme form of open PNP. Exencephaly was
considered to be present if the cranial neural tube was open, with or without
eversion of neural folds, in embryos containing 30 or more somites (by which
stage the cranial neural tube is probably closed in normal embryos).
The method used for analysis of PNP closure was as follows (see Fig. 1).
A small quantity of 1 % aqueous toluidine blue solution was injected into the
closed thoracolumbar portion of the neural tube using a mouth-controlled
glass micropipette (approximately 100/*m diameter). Dye usually spread
rostrally along the neural canal, and often filled the hindbrain ventricular
system. Caudally, dye was either extruded from the neural tube into the surrounding medium (classified as open PNP) or was confined within the caudal
portion of the neural canal (closed PNP). In embryos of the latter type it Was
unusual for dye to penetrate as far as the caudal tip of the neural canal, presumably because of increasing pressure in the closed canal. The possibility that
high intraluminal pressure exerted during dye injection may have led to
rupture of closed PNPs in some embryos, with consequent incorrect classification
of their PNP status, was investigated by histological analysis of the PNP region
in two types of embryos: (a) those appearing on visual examination to have
a closed PNP, but in which dye injection demonstrated a small open PNP
(7 embryos analysed: 1 A/Strong, 6 CTxCT), and (b) littermates of these
embryos, in which the PNP was closed as judged both by visual examination
154
A. J. C O P P , M. J. SELLER AND P. E. POLANI
Fig. 1. Day 12CTxCT embryo with tail flexion defect, undergoing injection of
dye into the neural canal as a test for PNP closure. Dye has spread rostrally into
the cervical region and caudally almost to the tail tip. Lack of dye extrusion into the
surrounding medium demonstrates that PNP closure is complete (x 20).
and dye injection (8 embryos analysed: 5 A/Strong, 3 C T x C T ) . Tail tips
were fixed for 4 h in cold 2-5% glutaraldehyde in phosphate buffer, washed
in cold phosphate buffer, postfixed in osmium tetroxide and embedded in
TAAB resin. Serial sections were cut at 2 /im, transverse to the long axis of
the tail, and stained with toluidine blue. Embryos classified by dye injection
as having a closed PNP were found in all cases to have a completely closed
caudal neural tube (Fig. 2 a, b, c), whereas all embryos classified by dye
injection as having an open PNP showed a small distal region of open neural
folds which showed no signs of having resulted from rupture of a closed
neural tube (Fig. 2d, e,f). It may be concluded that the dye injection technique
provides a more reliable and accurate method of assessing PNP status than
is possible by visual examination alone.
Assessment of developmental stage
The number of somites was utilized as a convenient measure of developmental stage. Counting somites was standardized in the following ways. Only
complete somites were counted. Somite 11 was consistently found to lie
opposite the caudal edge of the forelimb bud, and somites 24 and 28 opposite
i
g-ft>
*i «
«. -•
Fig. 2. Transverse plastic-embedded sections (2 /tm) through the PNP region of A/Strong and ct embryos following classification
of PNP status by dye injection* (A), (B) and (C); embryos with closed PNP as judged by both visual examination and dye injection.
Note the closed neural tube which was present in serial sections. (D), (E) and (F): embryos in which the PNP appeared closed on visual
examination, but in which dye injection demonstrated a small open PNP. Continuity between surface ectoderm (se) and neurectoderm (nt) in all three embryos indicates that the region of open neural folds is persistent and has not resulted from recent rupture of
a previously closed neural tube. The mean number of 2 /*m sections in which open neural folds were present in the seven embryos
analysed was 30-0 ± 17-4. Thus the PNP had an average axial length of 60 /*m. Sections stained with toluidine blue (x 360).
se
B
§
f
8-
156
A. J. COPP, M. J. SELLER AND P. E. POLANI
sir
,~f
Fig. 3. (A-D) Transverse sections through the 'ascending limb' of ct embryo tails
with flexion defects (A, in vivo; C, in vitro) and 'comparable sections' (see below)
through straight tails (B, in vivo: D, in vitro). Cross-sectional areas occupied by
neural tube (nt) and neural canal (nc), and the total cross-sectional area of the tail,
were measured from photographs using an electronic digitizer (Model 1224,
Numonics Corporation). The area of neurectodermal tissue was calculated by
subtracting nc area from nt area. All areas are expressed in Table 2 as proportions
of total tail cross-sectional area, in order to avoid spurious results due to variations
in size between embryos. Sections stained with haematoxylin and eosin (x 360).
the rostral and caudal edges of the hindlimb bud, respectively. Consequently
all embryos were checked against this 'standard somite pattern', to ensure
uniformity of the counting procedure. In a few embryos, where rostral somites
were not clearly visible, the 11th and 28th somites were used as landmarks
from which to begin counting. The reliability of the above method of somite
counting was independently checked by comparing the stage of lens develop-
Neural tube development in mutant and normal mouse embryos 157
(i)
(ii)
(iv)
(E) Diagram to illustrate the method used in locating 'comparable sections' for
analysis of cross-sectional areas, (ii) and (iv) are side views of straight and flexed
tails, respectively; (i) and (iii) are schematic transverse sections showing outlines of
tail and neural tube at axial levels, 1,2 and 3. Asterisks indicate a pair of'comparable
sections'. Since differences in cross-sectional area could arise from variations along
the length of tails, sections for comparison were matched in the sense that they
occurred at equal distances from the caudal end of their respective neural tubes
(defined here as the caudal level at which the neural canal no longer appears in
transverse sections). These distances were found, for straight tails, by counting
6 fim transverse sections (distance x), and for flexed tails by the sum of the lengths
of 'ascending' (a) and 'descending' (d) limbs, found by counting sections, and the
length of the 'horizontal' limb (h) in mid-sagittal section (at axial level 1) as
measured by an eyepiece graticule.
EMB
69
158
A. J. COPP, M. J. SELLER AND P. E. POLANI
ment with somite numbers in embryos which had developed either in vivo or
in vitro. Embryonic heads, fixed in Bouin's fluid, were embedded in paraffin
wax, serially sectioned in a frontal plane at 6/on, and stained with haematoxylin and eosin. Lens development was divided into stages as described by
Theiler (1972).
Morphological analysis of the curly tail defect
Tails of embryos from ct matings, which either exhibited curly tail flexion
defects or were straight (and which had 35 or more somites), were embedded
in paraffin wax, serially sectioned at 6 /*m, and stained with haematoxylin and
eosin. Straight tails were sectioned transversely to the long axis, whereas tails
with flexion defects were oriented so that the region immediately proximal to
the bend (the 'ascending limb') was represented in transverse sections. A
comparison of the proportion of tail cross-sectional area occupied by neural
tube, neural canal and neurectodermal tissue in straight tails and in tails
with flexion defects was made as shown in Fig. 3.
RESULTS
Posterior neuropore (PNP) closure in vivo and in vitro
The proportion of embryos with an open PNP, as judged by dye injection, is
plotted against somite number in Fig. 4. A/Strong, CTxCT, and STxST
embryos begin PNP closure in vivo (Fig. 4 a) between 30 and 34 somites, but
whereas more than 90% of A/Strong embryos have a closed PNP by 34
somites, and 100 % by 39 somites, the PNP in 57 % and 36 % of CT x CT and
ST x ST embryos, respectively, remains open. Moreover, even in the most
advanced C T x C T and STxST embryos examined (40-49 somites), about
20 % of embryos still maintain an open PNP.
Figure 4(b) shows the results of embryos developing from the headfold
stage in vitro. Onset of PNP closure is earlier (i.e. 25-29 somites) than in vivo
in all three categories of embryo, but whereas over 90 % of A/Strong embryos
have undergone PNP closure by 30-34 somites, the PNP remains open in
approximately 50 % of the mutant embryos at this stage. It was unusual for
embryos developing in vitro to reach stages with more than 40 somites.
Nevertheless, the results for ST x ST embryos at least indicate that a minority
of the most advanced mutant embryos maintain an open PNP in vitro as well
as in vivo.
No obvious differences were noted between CT x CT and ST x ST embryos
with regard to the stage of PNP closure either in vivo or in vitro.
Incidence of developmental defects
The results are shown in Table 1. A/Strong embryos did not develop tail
flexion defects, LSM or exencephaly either in vivo or in vitro. The incidence
Neural tube development in mutant and normal mouse embryos
159
10
0-8
06
0-4
0-2
15-19 20-24
25-29
30-34
35-39
40-44
45-49
-V 15-19 20-24
25-29
30-34
35-39
40-44
45-49
10
0-8
0-6
0-4
0-2
Somite number
Fig. 4. Graphs showing the relationship between proportion of embryos with an
open PNP and somite number in: (A) embryos which have developed entirely in
vivo; (B) embryos which have developed in vitro from the headfold stage. Continuous
lines represent embryos from A/Strong matings, dashed lines represent CT x CT
embryos, and dotted lines represent ST x ST embryos.
of tail flexion defects in mutant embryos in vivo is approximately 60% in
both CT x CT and ST x ST embryos. It is important to know whether embryos
which develop tail flexion defects are generally retarded in development in
comparison with their normally developing littermates. If this is so, then
delay of PNP closure in these embryos would assume less significance, and
could be seen merely as representing one aspect of a generalized developmental retardation. The majority of mutant litters in vivo, on day 12 of gestation,
contained embryos in the following phenotypic categories: (a) normal straight
6-2
160
A. J. COPP, M. J. SELLER AND P. E. POLANI
Table 1. Proportion of embryos from A/Strong, CTx CT and STx STmatings
which developed tail flexion and neural tube defects in vivo and in vitro*
Type of defect
Somite
number
1
A/Strong
CT xCT
A
A
STx ST
j
i
In vivo In vitro In vivo In vitro In vivo In vitro
000
000
000
000
000
0-15
0-50
000
0-42
0-00
0-26
0-71
0-52
000
0-60
0-64
000
0-61
014
000
35 or more 000
008
000
0-23
t(0/51) (0/13) (8/55) (0/19) (12/52) d/12)
000
000
30 or more
000
003
004
000
(0/75) (0/26) (2/75) (0/35) (3/72) (0/32)
Curly tail
30-34
flexion defect 35-39
40-44
45-49
Lumbo-sacral
myeloschisis
Exencephaly
* Many abnormal embryos manifested more than one type of defect. For the sake of
clarity, incidences are given independently in the Table. In vivo: LSM occurred alone in 2
cases (1 CTxCT, 1 STxST), combined with tail flexion defects in 17 cases (7CTxCT,
10 ST x ST) and combined with both tail flexion defect and exencephaly in 1 case (ST x ST).
The single case of LSM in vitro was combined with a tailflexiondefect. Exencephaly occurred
alone in 4 cases (2 CT x CT, 2 ST x ST), and combined with tail flexion defect and LSM, in
1 case.
t Parentheses indicate: (number of defective embryos/total number examined).
tailed embryos; (b) embryos with tail flexion defects; and (c) embryos with
tail flexion defects and LSM. Eleven of these litters (five CT x CT, six ST x ST)
were subjected to an analysis of variance (randomized block design) which
compared mean somite number (as a measure of developmental progression) of
embryos in each of the phenotypic categories ('blocks') and in each of the
eleven litters ('treatment groups'). Although litters varied significantly from
each other with respect to mean somite numbers of embryos (F = 3 0 1 ;
D.F. = 10, 20; P < 0 01), the phenotypic categories did not differ significantly
(F = 1-3; D.F. = 2, 20; P > 005). It is clear, therefore, that embryos with
tail flexion defects, with or without LSM, are not retarded with regard to
somite number in comparison with their straight-tailed littermates in vivo.
Table 1 shows that CT x CT and ST x ST embryos also developed tail
flexion defects in vitro, but at a lower frequency than in vivo. This is not
surprising in view of the fact that cultured embryos rarely developed as far
as stages at which tail flexion defects could usually be identified with certainty
(i.e. 40 or more somites). Nevertheless, the development of tail flexion defects
in 26 % and 42 %, respectively, of the most advanced CT x CT and ST x ST
in vitro embryos makes it seem likely that the developmental processes occurring in mutant embryos in vitro are a true counterpart of those occurring
in vivo.
The incidence of LSM in C T x C T and STxST embryos in vivo was 14%
and 23 % respectively (not significantly different; x2 = 1-28; D.F. = 1; P > 005).
Neural tube development in mutant and normal mouse embryos 161
Table 2. Proportion of total cross-sectional area occupied by neural tube, neural
canal and neural tissue in mutant embryos with flexed and straight tails
Neural tube area*
Total tail area
Embryo
pair no.
Flexed
tail
Straight
tail
1
2
3
4
5
6
0-22
0-19
0-23
0-36
0-23
0-18
015
005
0-21
008
008
0-17
7
8
9
10
016
016
0-18
0-30
009
0-13
010
013
Neural canal area*
Total tail area
Neural tissue areaf
Total tail area
"\
Straight
tail
Flexed
tail
Straight
tail
In vivo
0-10
0-11
012
0-22
0-12
008
004
002
007
001
001
005
012
008
011
0-14
011
010
Oil
004
0-14
007
006
012
In vitro
006
005
003
009
001
001
001
0-03
0-10
0-11
0-15
0-21
008
0-12
008
0-10
Flexed
tail
* Combined in vivo and in vitro values differ significantly (P < 0-01) between flexed
and straight tails (Wilcoxon Matched Pairs test).
t Combined in vivo and in vitro values do not differ significantly (P > 0-05) between
flexed and straight tails (Wilcoxon Matched Pairs test).
In contrast, LSM was only seen in one embryo in vitro, but the same explanation can be offered as for tail flexion defects, namely the difficulty in identifying with any certainty the presence of LSM in embryos with fewer than
40 somites.
Exencephaly was rarely seen in this study. Only five embryos exhibited the
defect in vivo (overall incidence of 3-4 %). Exencephaly was not seen in embryos
developing in vitro. Given an in vivo incidence of 3-4 %, 2 exencephalics would
be expected among the 67 in vitro embryos examined. This expectation is not
significantly different from the observed absence of exencephalics (x2 = 2-3;
D.F. = 1; P > 0 05).
Morphology of the curly tail flexion defect
There are very striking differences in morphology between comparable
transverse sections (see Materials and Methods) through straight tails and
through the 'ascending limb' of tails with flexion defects (Fig. 3). Table 2
shows that although the neural tube and neural canal each occupy a significantly
greater proportion of tail cross-sectional areas in flexed tails than in straight
tails (P < 001), there is no significant difference in the proportion of tail area
occupied by neurectodermal tissue (P > 0-05). Clearly, the neural canal is
162
A. J. C O P P , M. J. SELLER AND P. E. POLANI
Lens stage
No indentation
20-24
25-29
T
•••
ooo
T
D
Shallow indentation
O
DD
30-34
35-39
40-44
45-49
o
DDD
• •
OOO
D
T
•
ooo
QDDO
BIB
T
o
Deep indentation
T
ooo
Vesicle closure
ODD
T
Vesicle separation
• •
• •••
••
Fig. 5. Diagram showing the relationship between stages of lens development and
somite number in embryos of the following types: • , A/Strong in vivo; O, A/Strong
in vitro; • , CT x CT in vivo; D, CT x CT in vitro. Each symbol represents a single
embryo. T represents stages as quoted by Theiler (1972).
abnormally expanded in the region of the bend in flexed tails, but this is
unaccompanied by any increase in neural tissue. This conclusion depends on
the assumption that flexed and straight tails have total cross-sectional areas
which do not differ significantly. This is indeed the case (combined in vivo and
in vitro data: t = 0-69; D.F. = 9; P > 0 05).
Comparison of development in vivo and in vitro
The close resemblance between development in vivo and in vitro has already
been noted in the preceding sections. In vivo and in vitro embryos were
indistinguishable morphologically, except that embryos with more than 30
somites were slightly smaller in vitro than in vivo. The following, more
objective, assessments of embryonic development provide further support for
the similarity of embryos in vitro and in vivo:
(1) Figure 5 shows the stage of lens development reached by 68 embryos
plotted against somite number. There is a clear correlation between the two
developmental parameters reflecting the reliability of somite number as a
measure of developmental progression. In particular, it is noticeable that
embryos developing in vitro reached advanced stages of lens development at
the same somite numbers as in vivo.
(2) Figure 6 shows that the morphology of the developing lens in A/Strong
and CT x CT embryos was closely comparable in vivo and in vitro.
Neural tube development in mutant and normal mouse embryos 163
~A
'«%sA
Fig. 6. Frontal sections through embryonic heads which demonstrate the morphology
of the developing lens at the stage of vesicle closure in embryos of the following
types: (A) A/Strong in vivo; (B) CTxCT in vivo; (C) A/Strong in vitro; (D)
CT x CT in vitro. Abbreviations: lp = lensplacodejov = optic vesicle ;se = surface
ectoderm. Sections stained with haematoxylin and eosin (x 360).
(3) The mean gestational age (h pc) of advanced CT x CT embryos (35-39
somites) is only slightly greater in vitro (275-8 + 5-9) than in vivo (271-3 ±7*1;
t = 1-93; D.F. = 31; P > 005), indicating that development in culture occurs
at approximately the same rate as in vivo. The same is true for advanced ST x ST
embryos (in vitro, 273-7 + 6 0 ; in vivo, 265-9±10-7; t = 2-33; D.F. = 24;
0 02 < P < 0 05). The mean gestational age of advanced A/Strong embryos
shows much greater discrepancy (in vitro, 277-3 ±3-1; in vivo, 255-8 ±1-8;
t = 20-58; D.F. = 33; P < 0 01). This is due primarily to the slight advancement of development in. A/Strong embryos compared with mutant embryos of
the same gestational age. Whereas most mutant embryos with 35-39 somites
were contributed by litters analysed on the morning of day 12 of gestation
(270-275 h pc), A/Strong embryos had reached this stage in vivo by the evening
of day 11 (255-260 h pc). The slight retardation of development in culture, as
already noted, meant that A/Strong litters could not be analysed until the
morning of day 12, and therefore appeared more retarded than was actually the
case.
164
A. J. COPP, M. J. SELLER AND P. E. POLANI
DISCUSSION
In the Introduction, three questions were posed concerning the pathogenesis
of tail flexion defects in ct mice. These will be discussed in turn.
PNP closure and developmental defects in ct embryos
At a stage when PNP closure is almost complete in A/Strong embryos,
approximately half of all ct embryos maintain an open PNP (Fig. 4). Later
in development, when the majority of ct embryos have eventually undergone
PNP closure, around half of these embryos develop tail flexion defects in vivo,
and a smaller percentage in vitro (Table 1). These results provide independent
evidence in support of Gruneberg's (1954) suggestion that delay of PNP closure
is the main developmental lesion which leads to the appearance of tail flexion
defects. Further support comes from the demonstration that embryos with
flexion defects are not generally retarded in development in comparison with
their straight-tailed littermates. Thus, delay of PNP closure is probably a
specific developmental defect in these embryos.
A comparison of the incidences of developmental abnormalities in ct mice,
as found in the present experiments and in a previous study using the same
animal colony, carried out in 1976/1977 (Embury et al. 1979), leads to the
following conclusions:
(1) The incidence of curly tail flexion defects in embryos with more than
35 somites has increased (x2 = 7-5; D.F. = 1; P < 0 01), and no longer differs
between embryos derived from CT x CT and ST x ST matings (see Table 1;
incidences from Embury et al. 1979: CT x CT - 43 %; ST x ST - 31 %).
(2) LSM is marginally but not significantly (x2 = 2-6; D.F. = 1; P > 005)
more common in the present study (incidence from Embury et al. 1979:
CTxCT-11%).
(3) Exencephaly has shown a threefold decline in incidence (x2 = 4-38;
D.F. = 1; P < 005) in the present study (incidence from Embury et al. 1979:
8'7%).
It is possible that these changes have resulted either from genetic alteration
in the curly tail stock, due to inadvertent selection during breeding, or from
environmental factors such as alterations in the composition of diet or drinking
water, or in temperature of the animal rooms. It is unlikely that the changes
can be attributed merely to differences between the two studies in the criteria
used for identification of defects in embryos. An analysis of 22 newborn
litters, made concurrently with the present study, showed incidences of tail
flexion defects and LSM of 56-4 % and 20-9 % respectively (CT x CT matings)
and 56-4% and 16-1 % (ST x ST matings). Exencephalics were not seen at all.
These incidences do not differ significantly (P > 005) from those in Table 1.
Neural tube development in mutant and normal mouse embryos 165
Morphology of the curly tail flexion defect
Figure 3 shows that the neural tube is closed in the region of the curly tail
flexion defect. The structure of this region is abnormal, however, in comparison
with straight tails (Fig. 3; Table 2). The neural canal is locally over-expanded
and the neural tube is positioned on top of the mesodermal elements rather
than embedded within them. One interpretation of these observations is that
delayed PNP closure, together with continued normal development of structures
in the tail bud other than the neural folds (i.e. hindgut, notochord) impose
mechanical stresses on the developing trunk and tail. In the tail, although
PNP closure occurs, a residual flexion deformity persists. In the lumbosacral
region, the imbalance between rates of development of neural folds and nonneural structures is so great that in some cases neural fold closure is impossible
and LSM results. An alternative interpretation based on the neural tube
reopening hypothesis of Gardner (1973) should also be considered. As applied
to ct embryos, this might suggest that high intraluminal pressure in the neural
tube leads in many cases to local over expansion of the caudal neural canal
(due perhaps to some undefined weakness at this level), and in some cases to
rupture of the already closed neural tube and formation of LSM. The relative
merits of failure of closure versus reopening as explanations for the origin of
neural tube defects have been debated for many years (Warkany, 1971;
Gardner, 1973). The controversy is as yet unresolved and will require continuous
observations on individual embryos before an answer is likely to be obtained.
Development of ct defects in vitro
This paper represents the first account of the development of a genetically
induced malformation in mammalian embryos maintained in vitro. The results
indicate that:
(1) Curly tail defects develop independently of maternal uterine environmental influence, at least from the headfold stage onwards. A similar conclusion
has been reached from experiments in which ct embryos were shown to develop
defects at a similar frequency following transfer at the blastocyst stage to
uteri of either non-mutant or ct mice (Seller, Beck, Adinolfi & Polani, 1981).
(2) The occurrence in vitro of developmental events which culminate in
production of curly tail defects provides an opportunity for experimental
analysis of a genetically induced abnormality of neural tube development in
the mammalian embryo.
Posterior neuropore closure during normal development
Closure of the PNP occurred in more than 90 % of non-mutant embryos at
stages between 30 and 34 somites in the present study. This result is consistent
with descriptions of embryos presented by Theiler (1972). However, several
other accounts of mammalian development report that PNP closure occurs at
166
A. J. COPP, M. J. SELLER AND P. E. POLANI
a much earlier stage of development: for example, at 21-25 somites in the
CFI-S mouse strain (Rugh, 1968); 24 somites in the rat (Witschi, 1962); and
25 (Hamilton & Mossman, 1972) or 29 (Marin-Padilla, 1978) somites in man.
Species differences may account for some of this variation, but it seems unlikely
that the stage of PNP closure can vary by as much as 10 somites between
different mouse strains. Alternatively it is possible that analysis of PNP
closure by observation alone is not a reliable method. In the present experiments, each embryo was classified twice with regard to PNP closure: first by
visual examination and then, immediately, by dye injection. Results from the
two methods were in complete agreement for embryos with 15-19 somites, but
at higher somite numbers an increasing percentage of embryos was either
incorrectly classified by visual examination (assuming dye injection to be
relatively free from artifactual results; see Materials and Methods), or could
not be classified with any certainty. Somite categories around the stage of
observed PNP closure were associated with the following levels of discordant
classification: 25-29 somites-17%; 30-34 somites-21%; 35-39 somites 12%. It seems possible that previous determinations of the timing of PNP
closure, in rodent embryos at least, could have been subject to considerable
error. Failure to detect small persistent neuropores may have led to underestimation of the number of somites present at the time of PNP closure.
We thank Mr J. A. Crolla for valuable discussion, Miss H. Gray for technical assistance,
and Professor M. J. Moses for the use of his electronic digitizer. The work was supported
by a grant from the Mental Health Foundation.
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(Received 23 July 1981, revised 24 January 1982)