Download a new mutant gene causing multiple abnormalities in the mouse

/ . Embryol. exp. Morph. Vol. 17, 3, pp. 543-81, June 1967
With 3 plates
Printed in Great Britain
543
Extra-toes: a new mutant gene causing multiple
abnormalities in the mouse
ByD. R.JOHNSON
M.R.C. Experimental Genetics Research Unit,
Department of Animal Genetics, University College London
INTRODUCTION
The present paper deals with the genetics, morphology and developmental
anatomy of a new mutant gene in the mouse called extra-toes. Extra-toes
(symbol Xt) derives its name from the fact that in the heterozygous condition
there are extra digits on the preaxial side of both hind feet. Apart from this (and
corresponding changes in the forefeet) heterozygotes are externally normal. The
homozygote dies at birth or in utero with a wide range of abnormalities. These
include oedema, paddle-shaped feet with up to eight digits, hemimelia and gross
malformations of the brain, central nervous system and sense organs.
ORIGIN
Extra-toes arose in the control series of an irradiation experiment at the M.R.C.
Radiobiological Research Unit at Harwell. Five Xtj + <$$ and two Xt\ + $$ were
sent to the Animal Genetics Department at University College London in
November 1962. Two of the $$ were mated with the ??, and the remaining three
with $? from the CB stock (the F1 of CBA/Gr and C57BL/Gr). An Xt\ + $ was
outcrossed to two CB $$ in November 1963. With these exceptions the stock has
been brother/sister mated throughout.
GENETICS
Extra-toes is a semi-dominant gene with almost complete manifestation in the
heterozygote and few normal overlaps. All four feet are usually affected. It is
an embryonic or neonatal lethal when homozygous. This is reflected in the
segregation figures (Table 1). In backcross matings (1) the number of XtjXt
animals recovered was 45 whereas the expected number was 261 (i.e. XtjXt and
+ / + should be found in equal numbers). The numbers of Xt/+ and + / +
animals are close to the expected 2:1 ratio. In outcross matings (2) the figures are
in good agreement with the expected 1:1 ratio.
The deficiency of XtjXt animals in backcross matings together with the deAuthor's address: Department of Animal Genetics, University College, Gower Street,
London, W.C.I, U.K.
544
D. R. JOHNSON
creased litter size suggest that homozygous abnormals are lost before or at birth.
As Xt homozygotes can be identified from the 9th day of gestation onwards
viability may be studied directly. Table 2 gives details of 88 litters of embryos
recovered from backcross matings. The mean viability of XtjXt embryos is
0-72, suggesting that a number ofXt/Xt zygotes die before they can be recognized.
The number of homozygotes recovered at birth, however, is only 0-17 of the
expected total. The discrepancy between these figures reflects the nest tidying
activity of the mother, who often consumes stillborn young.
Lyon, Morris, Searle & Butler (1967) have shown that Xt is situated in
linkage group XIV, near to the gene for crinkled (cr).
Table 1. The segregation of Extra-toes at birth
Mating
type
1 Xt/+xXt/ +
2 Xt/+x+/ +
+ /+xXt/+
3 + / + x +/ +
/+
Xt/+
Xt/Xt
Unclassified
261
548
508
522
45
—
6
25
1055
21
—
—
—
21
Total
860
Mean
litter
size
4-9
7-2
70
Table 2. Segregation of Extra-toes in litters of embryos
Age
(days)
9
10
11
12
13
14-18
Total
Xt/ +
57
58
68
74
34
12
52
96
451
Xt/Xt
Solid
moles
Total
No. of
litters
Mean
litter
size
7-1
6-4
7-4
6-4
6-3
7-6
70
19
14
21
15
8
31
3
5
7
7
3
35
96
57
214
11
12
13
15
9
28
108
60
619
88
79
77
96
Viability
of Xt/Xt
0-97
0-72
0-92
0-61
0-52
0-63
0-72
MATERIAL AND METHODS
Xtj + $S and $$ were put together at 5 p.m. The females were examined for
vaginal plugs at 9 a.m. the next morning. The date of the vaginal plug was taken
as day 0 of gestation. Pregnant animals were killed with ether and their uteri
removed. If the pregnancy was in its 1 lth day or less the whole of the uterus was
fixed in Bouin's fluid and the embryos dissected out 24 h later. Older embryos
were dissected out in saline before fixation. Developmental age was checked
against the criteria of Griineberg (1943).
Sectioned material is listed in Table 3. It was not considered necessary to
section whole litters beyond the 9-day stage when homozygous abnormal animals can be readily identified externally. Therefore embryos were sectioned in
matched pairs or trios of litter-mates. All material was dehydrated and double
545
Extra-toes: a new mutant
embedded by the technique of Peterfi. Sections were cut at 5-10 jtc according to
age. The heads of newborn and older mice were decalcified in 4 % nitric acid in
90 % alcohol before dehydration and sectioned at 15 /i. All sectioned material
was stained in Ehrlich's haematoxylin and eosin.
The skeletons of extra-toes mice and their normal litter-mates were studied as
methylene blue and alizarin red clearance preparations (Table 4). Methylene
blue preparations were made by Noback's modification of Van Wijhe's method
according to Griineberg's (1953) protocol. Alizarin red preparations were made
by a modification of the technique of Dawson (1926).
Table 3. Serial sections of embryos and young mice
Xt/Xt
Unclassifiable
0
0
1
15
9
6
7
5
1
2
5
2
—
53
8
18
2
1
0
0
0
0
0
0
0
0
0
29
Age
(days)
Xt/ +
+ /+
0
0
2
19
14
7
8*
8#
9
9i
10
11
12
13
141
161
Newborn*
Newbornf
14 days post partum*
Total
* Heads only,
4
4
1
6
7
0
5
3
3
0
2
8
2
4
91
Total
8
18
5
35
23
13
14
12
2
10
20
4
9
173
f A series of excised organs.
Table 4. Materials used in the study oftheXt skeleton
Type of preparation
Methylene blue
Alizarin red
Age (days)
+ /+
Xt/ +
Xt/Xt
Total
15
18
3
1
3
4
6
1
7
3
6
1
8
—
15
3
18
7
6
3
29
13
5
29
3
—
—
22
8
58
Newborn
4 days post
partum
Newborn
4 days post
partum
THE Xtj + MOUSE
External anatomy
The extra-toes heterozygote is fully viable and breeds freely. It is of the same
size and weight as its normal litter-mates: at birth it is slightly larger and heavier
546
D. R. JOHNSON
Table 5. Birthweights of 231 animals from the Xt stock
Xt/ +
1 Xt/+xXt/ +
2 Xt/+x+/+\
+ / + xXt/+)
No.
Mean wt. (g)
No.
Mean wt. (g)
48
1-365 ±0053
98
1-434 ±0-028
40
1-432 ±0-032
45
1-501 ±0051
"1 cm
Text-fig. 1. Plantar views of the feet of + / + and Xt/ + mice. A, + / + right forefoot;
B-D, Xt/+ right forefeet; E, + /+ right hind foot; F-L, Xt/+ right hind feet.
(Table 5). Classification is possible from birth onwards by the presence of extra
pre-axial digits on the hind feet.
Plantar views of the feet of mice from the Xt stock are shown in Text-figure 1.
The forefoot of the normal mouse (A) carries four clawed toes and a pollex
represented by a small knob of tissue bearing a nail. In Xt/ + mice (B-D) the
pollex is enlarged or duplicated. Digit II may carry a double claw. On the postaxial side of the foot a small lump often appears opposite the base of digit V,
which may also carry a double claw.
Extra-toes: a new mutant
547
In the hind foot (F-L) a sixth or even a seventh toe is added preaxially. The
form of the extra digit is variable. In low grades the hallux is simply thickened
and may or may not carry a double claw. Progressively more abnormal mice
have a duplicated hallux whose constituents are united by soft tissue syndactylism,
or two or three entirely separate toes. Neither postaxial nubbins nor duplications
of the claw of digit V has been observed in adults: the hind feet of newborn
Xt\ + mice, however, sometimes carry a post-axial rudiment which regresses in
the first few days of life.
Belly spots
The original Xtl + mice obtained from Harwell all had white belly spots. This
characteristic has persisted in the present Xt stock (Table 6). The largest belly
spots seen are roughly circular, approximately 1 cm in diameter and situated in
the midline. All grades between this and-a few white hairs only have been seen.
In some cases the spot is elongated to form a streak. One Xtl + <$ developed an
asymmetrical head blaze late in life, but apart from this no head dots have been
seen.
Table 6. Belly spotting in the Xt stock
Xt/ +
+ /+
Spot
No spot
Total
Spot
No spot
Total
184
59-2%
127
40-8%
311
7
2-9%
237
97-1%
244
Table 7. The size and occurrence of the interfrontal bone in Xt/ + mice
Size of interfrontal
bone
Xt/ +
0
+
+ +
15
1
5
11
9
8
+ +
0
9
+
Total
29
29
* m
j „
The axial skeleton
1. The skull
The only abnormality seen in the Xt/ + skull is the presence of an interfrontal bone (Truslove, 1952) in a high proportion of cases (Table 7). In normal
litter-mates an interfrontal was present less frequently; where both members of
a pair of litter-mates had an interfrontal bone it was larger in the Xtl + mouse in
all cases save one, where the bones were of equal size.
2. Hydrocephaly
Six hydrocephalic mice have been recorded in the Xt stock to date. All were
Xtl + a n d occurred in one family. Xt 52/55 produced 4 known hydrocephalics
in 43 offspring. Of the 39 non-hydrocephalics 3 pairs were brother/sister mated
and produced 1/25, 1/58 and 0/60 abnormals respectively.
548
D. R.JOHNSON
3. The vertebral column and thorax
A vertebral count of 29 Xtj + mice and their normal litter-mates shows that
the total number of presacral vertebrae in Xtj + and + / + mice is identical.
There is no shift in the lumbo-sacral border as seen in some other polydactylous
mutants. The ribs and sternebrae of Xtj + mice are normal.
The appendicular skeleton
1. The cartilaginous skeleton
In the normal embryo of 15 days the cartilaginous precursors of all the limb
bones can be seen (Text-fig. 2 A). There is some ossification of the long bones and
the limb girdles.
Text-fig. 2. Forelimbs of 15-day-old embryos. A, + / + right; B, Xtj+ right; C,
Xt/+ left. Camera-lucida drawings of methylene-blue clearance preparations.
In the forelimbs the Xt heterozygote shows a series of duplications of the first
digit and the first metacarpal (Text-fig. 2B, C). In more extreme cases digit II
may also be partially duplicated. The small supernumerary digits which sometimes appear at the base of digit V have a single rod-like skeletal element.
In the hind limb (Text-fig. 3) duplications of the phalanges and metatarsal of
digit I can be seen. The cartilaginous precursor of the incomplete metatarsal or
splint bone, which is usually found on the preaxial side of metatarsal II is also
present.
2. The osseous skeleton
The abnormalities of the osseous skeleton of the forelimbs of Xtj + mice do
not correspond exactly with the pattern seen in their cartilaginous precursors.
It is clear from examination of Text-figs. 4 and 5 and comparison with Text-fig. 2
that the situation has been complicated by tertiary distal fusion of the osseous
Extra-toes: a new mutant
549
elements on the preaxial side of the foot. In Text-fig. 5B, for example, fusion has
occurred between the claws of both parts of digit I and between this double
element and the claw of the preaxial part of digit II. As the anatomy of the forefoot becomes more abnormal these fusions make interpretation more difficult.
In Text-fig. 4B and D there is fusion of the Os centrale with the Os multangulum minus. Postaxial accessory ossifications (A, Text-fig. 4D) were present in
10 out of 29 preparations examined. In four cases they occurred bilaterally,
whilst six mice had a unilateral occurrence on the right side. The converse, with
the element present on the left side only, did not occur in the sample.
1 mm
Text-fig. 3. Left hind limbs of 15-day-old embryos. A, +/+;B,
Xt/+.
The lowest grade of abnormality seen in the hind feet of Xtj + mice is the
presence of a small splint of bone adjacent to the base of Os metatarsale II
(Text-fig. 6). This may be present alone in a foot which externally looks quite
normal or may be associated with other abnormalities. It may occur as a separate
entity or be fused with the Os tarsale primum or (less frequently) the Os metatarsale secundum. The Os tarsale primum is thickened in low grades of polydactyly and may bear a pre-hallux consisting of a single phalanx and claw;
alternatively the metatarsal may be completely duplicated (Text-fig. 7C). In
more extreme expressions it is the more medial of the two Ossa metacarpalia
prima which divides or tends to divide to form a seventh digit. The Os tarsale
tertium may be fused with the Os tibiale; this condition tends to be restricted
to feet with a high grade of polydactyly (Text-fig. 7B, C). No other fusions were
observed amongst the tarsalia.
Hind-foot polydactylism in Xtj+ is remarkably symmetrical (Table 8):
62 % of the animals classified were symmetrically affected, and in cases of
asymmetry the right exceeded the left as often as vice versa.
550
D. R. JOHNSON
3. Hemimelia in heterozygotes
This has been seen in the Xt stock upon two occasions. In the first case,
a $, there was total absence of the tibia and a reduced bowed fibula in the left
hind limb. In the foot digit I was absent. The right hind limb was normal with
five toes. The forefeet were typical of Xtj + mice, with preaxial polydactyly. The
C+tA-
Text-fig. 4. Left forefeet of + / + (A) and Xt/+ (B-D) mice. Extensor surface, 28
days old. Camera-lucida drawings of alizarin clearance preparations. A, Accessory
postaxial digit. C+ M, Fused centrale and multangulum minus.
Extra-toes: a new mutant
551
Table 8. Symmetry of hind foot polydactyly at weaning in Xt/+ mice
L only
L>R
L=R
R>L
R only
Total
3
0-45 %
100
17-4%
357
62-1%
113
19-6%
3
0-45%
576
0-25 cm
Text-fig. 5. Left forefeet of + /+ (A) and Xt/+ (B-D) mice. Lateral view.
vertebral column was abnormal with some wedge-shaped vertebrae and
fusions. There was marked scoliosis in the lumbar region and multiple fusions
of the sacral vertebrae. The sternum was twisted, with irregular rib insertions.
Genetically the mouse proved to be Xtj +. Mated with an Xtj + brother she
produced 43 offspring (8 XtjXt, 24 Xtj + t 11 + / + ). None of the Xtj+ nor
+ / + were hemimelic. She was killed at 450 days, and an alizarin red clearance
preparation was made.
552
D. R. JOHNSON
The second case (from a mating between heterozygotes) was stillborn, and
was stained with methylene blue. In the left hind limb the tibia was reduced and
the fibula bowed. Digit I was triphalangeous with a reduced metatarsal. The skin
s+o
Text-fig. 6. Left hind feet of + / + (A) and Xt/+ (B-D). mice. Extensor surface
28 days old. S, Splint bone; S+O, splint fused to Os tarsale primum.
of digits I and II was united distally. The other hind limb was polydactylous, and
the forefeet typical of Xtj +. The vertebral column was abnormal throughout with
wedge vertebrae and fusions. The tail was kinked in several places. The ribs were
Extra-toes: a new mutant
553
not inserted opposite each other on the sternum. The parents produced twentythree other offspring, none of which was hemimelic.
0+ T
Text-fig. 7. The left (A, B) and right (C, D) hind feet of Xt/ + mice. O + T, Fused Os
tibiale and Os tarsale tertium.
THE XtjXt MOUSE
External anatomy
Perhaps the most striking characteristic of the newborn XtjXt mouse, apart
from its grossly abnormal limbs, is the retention of embryonic posture (Textfig. 8). The normal mouse embryo has lost the C-shaped curve of its vertebral
35
J EE M 17
554
D. R. JOHNSON
column by the 18th day of gestation. The column then becomes S-shaped by a
flexure appearing in the cervical region. This is even more pronounced at birth.
In the extra-toes homozygote, however, the C-shape is retained and even
accentuated. There is often a parietal brain hernia and ectopia of the
viscera. The skin is less wrinkled than normal. The limbs carry eight or nine
distinct toes, always webbed. Supernumerary papillae on the forehead carry
hairs. The abnormal arrangement of the mystacial vibrissae is shown in Textfigure 9.
One or two animals with neither pronounced ectopia nor protruding brains
have been born alive. They suffered respiratory distress and lived for only a few
minutes.
5 mm
2 mm
Fig. 8
Fig. 9
Text-fig. 8. Camera-lucida drawings of newborn mice. Left, + / + ; Right, Xt/Xt.
Left fore- and hind-feet of Xt/+ litter-mate.
Text-fig. 9. Arrangement of mystacial vibrissae in newborn + / + (left) and Xt/Xt mice.
The viscera
Dissection of six newborn homozygotes (2 $$, 4 $$) revealed the following
visceral abnormalities. The adrenal glands were displaced caudal so as to lie
medial to the kidney. In one female the left adrenal gland was double.
In all four females the right ovary was attached to the lateral side of the
right kidney, whence the oviduct/uterus ran normally. In two animals the
left ovary lay beside the left adrenal gland. Males were not correspondingly
affected.
In all cases the kidneys were irregular in outline with depressions seemingly
caused by the close proximity of other organs. For example, all kidneys had
indentations in the region lying adjacent to the bladder.
The following organs were removed from newborn homozygotes, sectioned
555
Extra-toes: a new mutant
and compared to normal controls: adrenal gland, gonad, heart, kidney, liver,
lung, spleen, stomach and thymus. No histological abnormalities were detected.
The brain
A single glance is enough to show the gross abnormality of the XtjXt brain
(Text-figs. 10,11). In dorsal view the whole brain is twisted about its major axis.
The olfactory lobes are absent. The cerebral hemispheres are small and partially
C£
Fig. 10
Fig. 11
Text-fig. 10. Dorsal and ventral views of the brains of newborn mice. A, Normal
dorsal; B, Xt/Xt dorsal; C, normal ventral; D, Xt/Xt ventral. CE, cerebellum; CH,
cerebral hemisphere; CQ, corpora quadrigemina; D, diencephalon; IN, infundibulum; MO, medulla oblongata; Nl, olfactory nerve; Nil, optic nerve; OL, olfactory
lobe.
Text-fig. 11. Lateral views of the brains of newborn mice. A, Normal; B, Xt/Xt. For
abbreviations see Text-fig. 10.
hidden beneath the bulging mesencephalon. The pineal body is missing; the
corpora quadrigemina are approximately normal in size but abnormally divided
laterally. The medulla oblongata is twisted and the cerebellum retains its undifferentiated folded form. Ventrally the optic chiasma is ill-defined (or absent in
35-2
556
D. R. JOHNSON
some cases). It is clear from Text-fig. 11 that the first flexure of the brain which
normally runs forwards at an angle is almost vertical in abnormal animals,
dividing the brain into two distinct parts.
Sections confirm the observations made externally. The cerebral hemispheres
contain groups of pycnotic cells. They are separated by a median fluid-filled
space which extends back as far as the cerebellum, and into which invaginates a
small, superficial choroid plexus.
The nose
The anterior part of the XtjXt nose is laterally compressed between abnormally
large maxillae and premaxillae (Plate 1, figs. A, B). The dorsal part of the nasal
chamber may be almost obliterated, and its ectodermal lining represented only
by a strand of tissue. The nasal septum is broad and short and the paraseptal
cartilages deformed.
The posterior nasal region, in contrast, is broad and flat (Plate 1, figs. C, D).
The structure of the nasal cartilages is more complex than usual. The olfactory
nerves are enormous. In parasagittal sections they can be seen to pass through the
cribriform plate; they then turn cephalad and terminate in a space filled with
connective tissue ahead of the cerebral hemispheres. The only contact between
brain and olfactory organ is by a very few fibres which turn caudad at the cribriform plate and enter the brain individually.
The nasal epithelium itself is abnormal. In Xtj + animals it is folded in some
areas, overlying a thickened lamina propria. In the homozygote the process is
extended; the epithelium is thrown into a series of folds which often raise it
considerably from the underlying cartilage.
The eye
At birth the eye of homozygotes may be up to half the normal diameter. The
eyelids are always closed. In more extreme cases the eyes may be represented by
a pigmented hollow ball containing a lens rudiment (Plate 1,fig.H), by a pigment
E X P L A N A T I O N OF PLATES
Abbreviations. AER, Apical ectodermal ridge; CE, cerebellum; CR, crus commune; ED,
endolymphatic duct; F, subarcuate fossa; /, upper incisor; L, lens; LC, lateral semicircular
canal; LSO, sense organ of lateral semicircular canal; M, maxilla; MAC, utricular macula;
N, nasal cavity; OE, oedematous area; OL, olfactory lobe of brain; ON, olfactory nerve; P,
pigment mass; R, retina; SC, superior semicircular canal; UT, utriculus.
PLATE 1
Figs. A, B. Transverse sections through the anterior part of the noses of normal (A) and Xt/Xt
(B) newborn mice.
Figs. C, D. Transverse sections through the posterior nasal regions of normal (C) and Xt/Xt
(D) newborn mice.
Figs. E, F, G, H. Left eyes of newborn mice. Transverse sections. E, + /+ ; F, Xt/+ ; G, H,
Xt/Xt.
J. Embryo/, exp. Morph., Vol. 17, Part 3
D. R. JOHNSON
J. Embryol. exp. Morph., Vol. 17, Part 3
D. R. JOHNSON
PLATE 2
Extra-toes: a new mutant
557
mass, or they may have disappeared without trace. Compensatory hyperplasia
of the Harderian glands helps to fill the orbit. When the eye is very much
reduced contact with the brain may be lost by atrophy of the optic nerve.
In three out of seven Xt\ + newborn heads sectioned the retinae of both eyes
were folded (Plate 1,fig.F). The folding was also recognizable in better developed
Xt/Xt eyes (Plate 1, fig. G). The retina here is very thick and surrounds the lens
in all sections, there being no pupil.
The ear
Of the three semi-circular canals of the normal labyrinth the posterior is best
represented in Xt/Xt (Plate 2, fig. J). It is normal in position and although it may
be small in diameter has a lumen throughout. The superior canal has a lumen only
posteriorly, where it leaves the crus commune, and anteriorly where it joins the
ampulla. Between those points it is seen only as a solid strand of tissue or as a
non-cartilaginous region of the capsule. The lateral semicircular canal is absent
in all cases, being represented only by its sense organ which is abnormally
situated adjacent to the utricular macula (Plate 2, fig. L).
* rr, , „
The axial skeleton
1. The skull
The skulls of XtjXt mice are always abnormal to a high degree. The normal
chondrocranium at 15 days is shown in Text-fig. 12 A. The method of closure of
the foramen opticum differs from that described by Griineberg (1953). This seems
to be a minor variant of the genetic background, occurring in the majority of
chondrocrania examined.
In Xt\Xt embryos (Text-fig. 12B) there is a complete division into anterior and
posterior parts by the failure of the hyphophysial cartilage and the trabecular
region of the central stem to unite. In the posterior part of the chondrocranium
the otic capsules are poorly differentiated. The occipital cartilages are heavy and
have an indistinct boundary over the foramen magnum. Anteriorly the alae
orbitales are heavy and help to form a broad pan upon which rest the cerebral
hemispheres of the abnormal brain.
2. The cervical vertebrae
In Xt\Xt mice there is a tendency for the neural arches of the cervical vertebrae
to fuse with their neighbours (Text-fig. 13). In B this has happened in three places
and the spatulate nature of the unfused neural arch rudiments is apparent. In C
PLATE 2
Figs. I, J. Transverse sections through the ears of newborn normal (I) and Xt/Xt (J) mice at the
level of the subarcuate fossa. Figs. K, L. Transverse sections through the ears of newborn
normal (K) and Xt/Xt (L) mice at the level of the utricular macula.
558
D. R. JOHNSON
almost all of the cervical vertebrae are involved and each side of the neck forms a
solid ridge. The vertebral bodies in all cases are quite normal with no signs of
fusion.
2 mm
Text-fig. 12. Dorsal views of the chondrocranium of a normal (A) and an XtjXt
embryo (B), 15 days old. A, Ala orbitalis; FM, foramen magnum; FO, foramen
opticum; M, Meckel's cartilage; O, otic capsule; P, parachordal cartilage; PA,
processus alaris; PN, paranasal cartilage; PT, parieto-tectal cartilage; 5", supraoccipital cartilage.
o
323
tSSS
V '•' "
'l''*
1 mm
Text-fig. 13. Dorsal views of the cervical vertebrae of a normal (A) and two Xt/Xt
embryos (B, C) 15 days old.
3. The sternum
Text-fig. 14 shows the ventral aspect of the thorax of a normal mouse and two
XtjXt litter-mates. Their developmental age is a little over 15 days as the sternal
Extra-toes: a new mutant
559
bands of the normal have united in the midline. In the abnormals, however, they
are still widely separated over most of their length.
4. The sacrum
In the XtjXt sacrum (Text-fig. 15) the situation is similar to that present in the
cervical region: there is a tendency for vertebral arches to fuse with their neighbours rather than with their fellows on the opposite side of the midline.
Text-fig. 14. Thorax of a normal 15-day-old mouse (A) and two XtjXt litter-mates (B, C).
1 mm
Text-fig. 15. Dorsal views of the sacral vertebrae of a normal (A) and an Xt/Xt embryo
(B), 15 days old.
The appendicular skeleton
The fore-and hind-limbs of XtjXt embryos show multiple abnormalities
(Text-figs, 16, 17). The number of complete digits rises as high as eight or nine.
The metacarpals and metatarsals may split distally or be represented only at
their distal extremities. The terminal phalanx of any finger or toe is often wholly
or partially duplicated, or absent. The separation between digits is less complete
than usual and the digits may be webbed. There is a variable amount of fusion
560
D. R. JOHNSON
amongst the carpals and tarsals, so much so that it is often difficult to identify
individual elements with any certainty. It is usual for one or more carpal or
tarsal to be united with a corresponding metacarpal or metatarsal by a narrow
isthmus of cartilage.
Text-fig. 16. Forelimbs of 15-day-old XtjXt embryos. A, Right; B, C, left. F, Foramen in fossa infraspinata; 0, ossified area.
Text-fig. 17. Hind limbs of 15-day-old Xt/Xt embryos. A, B, Right; C, left.
In the forelimbs the radius is occasionally represented by a proximal rudiment which may be no more than a spherical knob of bone adjacent to the
humerus, or extend half-way down the ulna. As a result of this hemimelia the
forelimb is more or less bent at the wrist so that the middle digits point across the
midline of the body. Usually, however, the radius is present and unreduced. The
humerus is short and thick; the crista deltoidea is large and coarse or absent.
Extra-toes: a new mutant
561'
The scapula often has a foramen in the fossa infraspinata or an indentation upon
its cervical margin. The acromion may be reduced or bowed.
In the hind limbs bilateral defect of the tibia is the rule. This, like the radius, is
then represented by a proximal knob of bone. The fibula is somewhat bowed in
extreme cases. The femur is short and stout. In the pelvic girdle the Os pubis may
be distorted forming a re-entrant in the outline of the foramen obturatum.
Hemimelia, when present, seems to be biased neither to left nor right (Table 9).
Ossification is retarded throughout the limbs of XtjXt embryos.
Table 9. Incidence of hemimelia in 23 Xt/Xt embryos
Forelimbs
Hind limbs
Hemimelia
absent
Left only
Right only
Bilateral
Total
19
0
3
1
1
1
0
21
23
23
1 mm
Text-fig. 18. Dorsal views of normal (left) and Xt/Xt embryos 9£ days old.
EMBRYOLOGY
It has not been possible to classify Xt/Xt embryos in segregating litters less
than 9 days old, either externally or from sections. At 9 days the homozygote can
be identified on the basis of two consistent abnormalities, a tendency for the
neural tube to remain open and the overgrowth of the first pharyngeal arch.
9\ days
At this stage the neural tube of Xt/Xt embryos (identified by their abnormal
pharyngeal region) may still be open from the otic vesicle forwards, sometimes
-562
D. R.JOHNSON
as far as the anterior neuropore. If the neural tube is closed (Text-fig. 18) the
roof of the rhombencephalon is abnormal, looking like the spade of playing
cards rather than being kite-shaped. Behind the otic vesicle the neural tube is
wavy to the level of the forelimb bud. The forebrain is pointed rather than
Text-fig. 19. Lateral views of the embryos drawn in Text-fig. 18.
1 mm
Text-fig. 20. Dorsal views of the limb-buds of 9-day-old embryos. Left, normal;
right, Xt/Xt.
rounded (Text-fig. 19) and the eyes may be elliptical rather than circular in outline, the major axis of the ellipse passing through the nasal region. The otic
vesicles are situated more dorsally than in normal litter-mates, and are thus very
close to the neural tube. They may still be open to the exterior. The pharyngeal
arches are enlarged, especially the mandibular arch which is directed laterally.
The anterior limb-buds present an abnormal outline when viewed dorsally
(Text-fig. 20); they appear lobed, adjacent lobes being separated by a slight
fissure.
In transverse sections (Text-fig. 21) the abnormal shape of the forepart of the
head can be seen to be due to the failure of the forebrain to expand. In the
563
Extra-toes: a new mutant
normal 9-day embryo the telencephalon is oval in cross-section; in Xt/Xt
animals its walls are closely approximated. The optic vesicles are situated with
their antero-posterior axis parallel to the overlying ectoderm and are thus not
orientated parallel to the midline. The lumen of the optic stalk is constricted.
The rhombencephalon, in this case, is widely opened.
OS mm
Text-fig. 21. Transverse sections through the region of the optic vesicles of 9-day-old
embryos. A, Normal; B, Xt/Xt. Projection drawings. O, Optic vesicle; T, telencephalon.
In frontal sections the rhombomeres are clearly visible (Text fig. 22). In
XtjXt embryos they are much enlarged, with the exception of the fifth which
seems to be constrained between the otic vessels. This is confirmed by reference
to transverse sections at this level (Text-fig. 23) where the neural tube is always
closed and lacks a roof plate. The anterior cardinal veins are somewhat enlarged and run lateral to the otic vesicles, rather than ventro-laterally.
10 days
At 10 days the external nasal processes of XtjXt embryos are reduced and the
forehead lacks the re-entrant normally seen in side view (Text-fig. 24B, C). The
first pharyngeal arch has differentiated into a maxillary and a mandibular portion;
the former is much enlarged. The pericardium is swollen and usually displaced.
The forelimb buds no longer show the lobes present at 9 days and no correspond-
564
D. R. JOHNSON
ing abnormality of the hind limb has been seen. The hind brain may still be open
(Text-fig. 24C) or may have been closed by an extensive roof (Text-fig. 24B).
In transverse sections of the normal 10-day embryo (Text-fig. 25) the telencephalic vesicles are more marked than at 9 days; the optic vesicle has become
an optic cup and lens induction is in progress. In the XtjXt embryo there is a
B
0-5 mm
Text-fig. 22. Frontal sections through the rhombencephalon of 9-day-old embryos.
A, Normal; B, Xt/Xt. Ot, Otic vesicle; R, rhombomeres; G, cranial ganglia.
bare suggestion of telencephalic vesicles. The wall of the prosencephalon between
these and the optic cup is folded; the optic vesicles are still flattened and their
axes displaced. Lens induction occurs only posteriorly; the rest of the optic
vesicle is no longer in contact with the overlying ectoderm. The nasal placode is
more extensive than normal, but invagination occurs only near its posterior end.
In the forelimbs the apical ectodermal ridges are increased in size (Text-fig. 26;
Plate 3, figs. M, N).
Extra-toes: a new mutant
565
0-5 mm
Text-fig. 23. Transverse sections through the region of the otic vesicles of 9-day-old
embryos. A, Normal; B, Xt/Xt. Ot, Otic vesicle; AC, anterior cardinal vein.
Text-fig. 24. Lateral and dorsal views of the heads of 10-day-old embryos. A, + /+ ;
B, Xt/Xt; C, pseudencephalic XtjXt.
566
D. R. JOHNSON
11 days
By the 11th day all surviving Xt homozygotes have performed some kind of
hind-brain closure, although this is always abnormal (Text-fig. 27). There is
still no invagination between the putative cerebral hemispheres. The optic
stalks have broad lumina and carry small optic cups, which usually have folds
Text-fig. 25. Transverse sections through the region of the optic vesicle of 10-day-old
embryos. A, + /+ ; B, Xt/Xt. Abbreviations as in Text-fig. 21.
II
0-25 mm
Text-fig. 26. Projection drawings of the AER of a normal (A) and an Xt/Xt (B) embryo.
11 days old. Dorso-ventral sections 7-5 ji thick. Every 5th section drawn, the middle
section of the five being the central section of the limb.
J. Embryol. exp. Morph., Vol. 17, Part 3
PLATE 3
J
D. R. JOHNSON
facing p. 567
Extra-toes: a new mutant
567
in their outer walls. In some embryos the eye cup has lost contact with the ectoderm and is filled with mesoderm.
The maxillary process, having grown out laterally, starts to extend forwards;
the ectoderm of its anterior face is thicker than normal.
Text-fig. 27. Transverse sections through the region of the optic vesicle of 11-day-old
mouse embryos. A, + /+ ; B, Xt/Xt. F, Fold in wall of optic vesicle.
12 days
In the homozygous abnormal there is a distinct bulge in the mesencephalic
region. The roof of the mesencephalon stands proud of the surrounding tissues,
covered only by a layer of ectoderm. Behind the otic vesicles the neural tube is
irregular with folded walls (Text-fig. 28).
In parasagittal sections of embryos of this age (Plate 3, figs. O, P) the developing nasal region is abnormal. Normally the roof of the nasal chamber is arched.
PLATE 3
Figs. M, N. Transverse sections through the anterior limb-buds of 11-day-old normal (M)
and Xt/Xt (N) embryos.
Figs. O, P. Parasagittal sections through the nasal regions of 13-day-old normal (O) and
Xt/Xt (P) embryos. The roof of the nasal chamber is arrowed in each case.
Figs. Q, R. Transverse sections through the trunks of 13-day-old normal (Q) and Xt/Xt
(R) embryos at the level of the umbilical hernia.
Figs S, T. Transverse sections through the ear regions of 13-day-old normal (S) and Xt/Xt
(T) embryos. The vertical axis is arrowed in each case.
568
D. R. JOHNSON
ooa
aatuo
i
i
1 mm
Text-fig. 28. Transverse sections at 100/* intervals through the neural tube of a
12-day-old Xt/Xt embryo. The first section is at the level of the second pharyngeal
arch, the last at the level of the anterior limb bud.
Text-fig. 29. Outline drawings of the limb buds of 12-day-old embryos. Camera
lucida drawings. A, + / + ; B, Xt/+ ; C, Xt/Xt right forefeet. D, + / + ; E, Xt/+,
F, Xt/Xt right hind feet.
Extra-toes: a new mutant
569
In Xt homozygotes, however, it is much flatter, and the nasal chamber thus
partially occluded. The olfactory nerve is less well developed than in normal
embryos of this age.
The eyes are reduced and rotated ventrally so that they look downwards into
the cheek. In some cases the maxillary process has grown forward over the eye
so that the latter is partially or completely obscured.
Text-fig. 30. Superimposed outline drawings of limb-buds; outlines as in Text-fig. 29.
Solid line, normal; dotted line, abnormal. A, Right forefoot, Xt/ + on + /+ ; C, right
hind foot, Xt/ + on + /+ ; B, Right forefoot, XtjXt on + /+ ; D, right hind foot
Xt/Xt on + / + .
At this stage the heterozygote can first be recognized by its footplates (Textfig. 29). The normal 12 day footplate has an indented outline, and regions
corresponding to five digits may be recognized. In the Xt\ + limb-bud the area
destined to become digit I is enlarged. The XtjXt limb-bud is much wider than
normal. The indentations between digits are less well marked. There is obvious
preaxial enlargement and superimposition of the outlines of normal and XtjXt
limb-buds (Text-fig. 30) shows that there is postaxial enlargement also. In the
hind limb the picture is similar. The Xt/Xt footplate has a pre- and a postaxial
widening, which seems to have occurred at the expense of proximo-distal
elongation.
13 days
The 13-day XtjXt embryo has developed considerable subcutaneous oedema
(Text-fig. 31). This takes the form of a swelling on either side of the neural tube
which extends from the cervical region to the root of the tail and laterally as far as
the proximal part of the limbs. In sections (Plate 3, figs. Q, R) it can be seen that
the oedema is due to expansion of the subdermal mesoderm which is less dense
than normal. In the thoracic region a second oedematous area is seen between
the lungs and the ribs. There is an enlarged umbilical hernia which includes
part of the liver. Mild oedema is sometimes evident in heterozygotes (8 of
34 examined) and in one case a clear fluid-filled bleb was present in the shoulder
36
J E E M 17
570
D. R. JOHNSON
region. The homozygote is given a hunch-backed appearance by its distended
hind brain. On one or two occasions a bleb was seen surmounting this hump.
The forehead and nose have an irregular array of epidermal papillae resembling
the follicles of sensory hairs, which in fact they represent. Up to 10 have been
seen, but 2 or 3 is more usual. The supra-orbital papillae are often displaced and
may be three rather than two in number. By contrast the post-orbital papillae
were reduced or absent in 17 out of 28 heads examined. The maxillary region is
enlarged and carries more follicles (which give rise to mystacial vibrissae) than
is usual. Normally five rows are visible; in XtfXt a partial sixth row is often
inserted and the number of follicles per row increased.
2 mm
Text-fig. 31. Camera-lucida drawings of + /+ (left) and XtjXt 13-day-old embryos.
The number of nipples visible in XtjXt mice at this stage is reduced from four
to two pairs, the most anterior and posterior ones persisting. Plate 3, figs. S, T,
shows transverse sections through the otic regions of normal and XtjXt embryos
respectively. In the normal there is an evagination in the wall of the presumptive utricular region opposite the point of entry of the endolymphatic
duct, which will later become the lateral semi-circular canal. In the abnormal
this evagination is absent. The seventh and the cochlear part of the eighth
cranial ganglia are enlarged.
14-16 days
The 14-day XtjXt embryo is still oedematous. The abdominal hernia has
reached an abnormally large size. Extra digits can be seen both pre- and postaxially on the feet of the heterozygotes, and some have an oval blood clot in the
frontal region of the head.
At 15 days the oedema is less marked but the skin appears taut all over the
body.
Extra-toes: a new mutant
571
At 16 days the mesencephalon may be exposed via an aperture in the cranium
which corresponds to the midcerebral bump found in younger animals. When
this occurs the amniotic fluid is always bloody and the embryo anaemic.
In sections of 14- to 16-day embryos small subcutaneous fluid-filled blebs
are seen, in locations likely to be overlooked on superficial examination. Such
blebs have been found on the tip of the lower jaw, on the underside of the upper
lip and in the external auditory meatus, at the base of the pinna. Heterozygotes
may also have blebs in the last position, and between the digits. Corneal blebs
are seen, in homozygotes only, when the eyes are much reduced and there is
partial premature closure of the eyelids.
DISCUSSION
The syndrome described in this paper is one of the most complex known from
the study of mammalian developmental genetics. In order to place the multiple
effects of the extra-toes gene in perspective it is necessary to understand the
actions and interactions of abnormal parts during development. From the many
abnormalities described above three groups of related disturbances emerge:
those due to (1) the maldevelopment of the nervous system, (2) the overgrowth
of the pharyngeal arches and limb buds, and (3) oedema.
The brain and CNS
The late closure of the neural tube is one of the earliest abnormalities seen in
Xt. The subsequent elongation of the forebrain and folding of the neural tube
point to an increase in volume of the neural tissue.
The forebrain, by failing to expand and continuing to elongate determines the
shape of the forepart of the head. The optic vesicles which are attached to the
forebrain must perforce be involved. The pointed forebrain leads to the apparent
decrease in the size of the nasal processes and probably to the abnormal invagination of the nasal placode. The failure of the roof of the telencephalon to
invaginate leads to the formation of small cerebral hemispheres abnormally
positioned with respect to each other. The anterior choroid plexus is formed very
late and is small and superficial. This may account for the pycnoses seen in the
cerebral hemispheres at birth.
Farther back, at the level of the otic vesicles, the roof of the neural tube is
less extensive than usual at the 9-day stage. Again this seems to be due to nonclosure of the forebrain, and consequent failure to expand owing to lack of
hydrostatic pressure. The folding of the neural tube is not uniform. It appears
first and reaches its greatest extent in three regions, cervically and at the thoracic
and lumbar flexures. This is the situation expected, if the folding is due to
overgrowth of neural tissue.
The neural tube may fail to close because of an intrinsic defect in the neural
tissue or an extrinsic factor which fails to allow the edges of the neural folds to
36-2
572
D. R. JOHNSON
meet and fuse. Examples of both mechanisms are found in the literature. In the
Loop-tail mouse Smith & Stein (1962) have suggested that the failure of the
neural folds to unite is due to the failure of the primitive streak (and hence the
neural plate) to elongate correctly. This is clearly an extrinsic dysraphism. In
Splotch, on the other hand, Auerbach (1954) considers that there is a primary
defect of the neural plate, and Smith & Stein suggest that the difficulty in closing
is due to an incomplete separation of the neural crest from the neural tube.
Patten (1952, 1953) has described a series of pseudencephalic embryos whose
brain defects are similar to those of extreme Xt homozygotes. He ascribes the
non-closure of the neural tube to overgrowth of neural tissue. Dekaban &
Bartelmez (1964), however, suggest that non-closure of the neural folds could
lead to an overgrowth of neural tissue. The work of Fowler (1953) supports
this hypothesis. She operated on chick embryos, slitting the roof of the neural
tube and thus producing artificial spina bifida. Embryos thus treated showed
neural overgrowth.
The eruption of the brain in the parietal region is considered to be a separate
event from the early failure of the neural tube to close. Exencephalic embryos
are not seen between 11 and 16 days. The inference is that those embryos seen at
16 days with exteriorized neural tissue have acquired it as a result of a secondary
disturbance acting upon the closed neural tube. Further, the protruding brain
is not turned inside out as in pseudencephalic mice, but simply protrudes
through the cranium. The Xt condition thus seems to resemble the mid-cerebral
lesions seen by Carter (1959) in my rather than the pseudencephalic mice
described by Bonnevie (1936) in the same stock.
, rrj
The sense organs
6
1. The nose
The first significant factor in the development of the XtjXt nose is the abnormal invagination of the nasal placode seen at the 10-day stage. At 12 days
the roof of the nasal chamber is flat rather than vaulted due to the excessive size
of the maxillary processes which form it. The posterior part of the nose is later
flattened by the abnormal brain, whilst its anterior region is compressed vertically by the huge maxillae. A little later still the forepart is further compressed
by the developing incisors. This may result in a loss of contact between the
nasal chamber and the exterior.
Another group of abnormalities is due to the excessive growth of the epithelium lining the nasal chamber, first visible at the 15-day stage. The olfactory
nerve consists of fibres which have their origin in the olfactory epithelium and
grow back towards the brain. At the 12-day stage this tract of fibres is poorly
developed in Xt\Xt\ at birth it is huge, notwithstanding the fact that contact
with the brain has not been made. It seems probable that the overgrowth of the
nasal epithelium gives rise to a correspondingly enlarged olfactory tract.
Extra-toes: a new mutant
573
2. The eye
The developing eye is first affected by the elongation of the forebrain at the
9-day stage. The proximal part of the optic stalk is carried forwards by the forebrain; the distal part with its attached optic vesicle lags behind. The latter is often
deformed by the force applied to it, and tends to become elliptical in crosssection. To a greater or lesser extent contact is lost between the surface of the
vesicle and the ectoderm. The eye which develops from such a vesicle must be of
reduced size and, if separation of the eye cup from the ectoderm continues, will
be invaded by mesenchyme cells.
The part of the optic vesicle not involved in lens induction, which is larger
than normal, is incorporated into the optic stalk or appears as a fold in the outer
layer of the eye cup. By the 12-day stage the developing eye is threatened further
by the maxillary process which grows over its lower part. Later still, at 15 days
the retina may start to proliferate and fold. Finally a corneal bleb may be present
in some cases. At a stage depending upon the net result of these variables the eye
ceases to develop further; hence in newborn XtjXt mice all stages are seen from
virtual absence of the eye to a small reasonably developed eye with a crystalline
lens and a folded retina.
3. The ear
The first sign of abnormality of the ear is the anomalous position of the otic
vesicles at the 9-day stage, probably due to slow development. From then
onwards they lie adjacent to an abnormal hind brain.
Deol (1964, 1966) has demonstrated the correlation between abnormal hind
brain and abnormal ear in a number of mutants. It seems that in Xt also the poor
differentiation of the ear is a direct result of the abnormal hind brain.
4. Oedema and blebs
The origin of the oedema which arises in XtjXt embryos at the 13-day stage is
not clear. A similar oedema in talpid3 mutant in the chicken was ascribed
by Ede & Kelly (1964a) to a mesodermal defect. In discussing the situation in
Patch, in the mouse, Griineberg & Truslove (1960) suggest that hydrops and blebs
'may be comparatively unspecific and rather far removed from the primary gene
action', pointing out that 'disturbances of liquid balance (oedema, blebs) are
often encountered in amphibian embryos which have been subjected to surgical interference.' On the other hand M. S. Deol (unpublished) has also pointed
out the association between blebs and neurological disturbances in the
mouse; Xt may well be yet another blebby/neurological mutant.
Whatever the origin of the oedema its results are clear cut. The force exerted
by the fluid contents of the swellings lateral to the spine disrupts the ventral
closure of the embryo, leading to umbilical hernia. The presence of oedematous
tissue in the dorsal part of the body leads to a reduction of space in the abdomen.
574
D. R. JOHNSON
The effects of the oedema are first on the sternum and secondly on the
viscera.
The abnormal position of the ovaries may be explained in the following way.
The mammalian gonad arises from the mesonephros, cephalad to the metanephros, and migrates caudad. The mechanism by which this migration is
achieved is quite different in the two sexes. In the male it is active, through the
contraction of the gubernaculum relative to the surrounding tissue. In the female
the ovaries migrate passively over a shorter distance under the influence of their
own weight. It is clear that in XtjXt males the process is normal, but that in the
females passive migration of the ovaries is hampered by cramped conditions in
the abdomen.
No direct sequelae to the presence of blebs in the form of haematoma or
thrombi of the type found in my and Ph have so far been discovered in Xt. However, the blebs shown by Xt homozygotes are much smaller and less widespread
in distribution than those present in conjunction with the other two genes.
5. Vibrissae
The number of primary and secondary vibrissae in the mouse is an almost
invariant character. Fraser & Kindred (1960) worked on the sex-linked gene
Tabby which affects the coat and reduces the number of secondary vibrissae.
After intensive selection they were able to produce lines of mice where the number
of vibrissae was greater or smaller than normal. When extra vibrissae were
present they were always limited to a pre-existing site, e.g. the number of supraorbital sinus hairs was sometimes increased from two to three.
Extra-toes shows abnormalities in the number of mystacial and supra-orbital
sinus hairs and in their pattern. There are also extra vibrissae present on the
dorsal part of the snout and on the forehead. There are therefore two elements
at work. One of these is the interference with the number of vibrissae at an
established site, as noted by Fraser & Kindred, and the other is the induction of
vibrissae at a site where none normally exist. The site in this instance includes
practically the whole of the forehead, with no recognized preference for specific
areas.
The skeletal system
The morphology of the skeletal system is largely subordinate to that of surrounding tissues. All the anomalies of the skeleton of XtjXt and Xtj + mice can
be related to pre-existing abnormalities in other structures.
The limbs
Polydactylism and hemimelia seem to be consequences of abnormal development of the limb-buds. The differences seen between the anterior limb-buds in
XtjXt and normal embryos predates the first limb-bud abnormality seen in any
other mutant (Griineberg, 1961) by 2 days. It is probable that a more detailed
Extra-toes: a new mutant
575
study of the early development of Xt limb-buds will reveal further changes in
structure.
The work carried out so far sheas no light on the controversial roles of mesoderm and ectoderm in limb development. It can only be stated that the distribution of mesoderm at 9 days is abnormal and that the AER is larger than normal
at 10 days, when this structure approaches its maximum development.
The polydactyly which occurs in Xt cannot be classified with relation to the
limb axis in the usual way as both pre- and postaxial digits are regularly added
even in the heterozygote. That the former are always larger in size and greater in
number than the latter is perhaps due to the postaxial-preaxial sequence of
mesodermal condensations in the footplate (Forsthoefel, 1963; Milaire, 1965).
However, the type of polydactyly found in the heterozygote accords quite well
with previous descriptions of polydactylous limbs.
Polydactyly seems to be divided into three morphological types. In the first
(e.g. Duplicate in the chick, Warren, 1941; Strong's luxoid in the mouse, Strong,
1961), part of a mirror image hand is formed, the digital sequence being V, IV,
III, II, I-I, II, III, etc. New elements appear preaxially to all existing material
in each case, and the hallux remains more or less normal. This type of polydactyly may be explained by postulating a splitting of the AER into two parts.
In the second type (which includes luxate (Carter, 1951) and fidget (Truslove,
1956)) and is characterized by the presence of incomplete metatarsals, the digital
sequence is less clear. Here digits are not added to the preaxial side of the foot
but arise from the splitting of digit I or II, even in the presence of a pre-hallux.
It is suggested that the organizing centre of the AER is enlarged but intact in this
case. The polydactyly of extra-toes heterozygotes is basically of this type, if the
post-axial digit is ignored.
The third type of abnormality is that shown uniquely by eudiplopodia in the
chick (Rosenblatt, Kreutziger & Taylor, 1959) where two-plane polydactyly is
correlated with the appearance of two separate AER's.
The hemimelia in Xt homozygotes could be explained following Forsthoefel
(1959). He suggested that the association of preaxial polydactyly and hemimelia
might be due to blastemal competition. The anterior portion of the footplate may
be able to 'take over' mesoderm from the anterior side of the forelimb anlage
for digit formation, thus leaving the radial region deficient: this is expressed
later as hemimelia. This argument, however, does not account for the presence
of hemimelia associated with ectrodactyly, as in Dh (Searle, 1964) and occasional
Xt heterozygotes, where there is a preaxial tissue deficiency in both limb and
foot.
The involvement of the limb-girdles is of a type previously described in relation to luxoid mutants. The presence of a foramen in the fossa infraspinata has
been described by Curry (1959) in droopy-ear and by Searle (1964) in postaxial
hemimelia (px). Curry showed that this defect is present in the membranous
skeleton and is therefore the result of a blastemal abnormality. Inpx the foramen
576
D. R. JOHNSON
probably dates from the same stage, and is certainly present in the cartilaginous
scapula. In XtjXt the Os pubis may be reduced or bowed. This is also seen in
Dh, 1st and Ix (Carter, 1951; Strong & Hardy, 1956; Strong, 1961; Searle, 1964).
These defects of the limb-girdles may be attributed to the redistribution of
skeletogenic material in the limb-bud.
The extensive bony fusions]of the digits on the preaxial side of the forelimb are
clearly tertiary in nature. No fusion of cartilaginous elements has been seen in
this region up to birth. During postnatal growth the proportions of the forefoot
change quite markedly, with the central digits elongating much more rapidly than
the first, which is rudimentary in the adult. It seems that during this period
the extra elements in the region of digit I become compressed and that fusion
takes place amongst them.
The two Xt\-v hemimelic animals which have occurred in the stock most
probably represent a rare variation of the Xt genotype; as both these mice were
descended from the outcross mating Xt5/CB 1125 it seems likely that the hemimelic form arose due to a change in genetic background.
2. The skull and axial skeleton
The abnormalities of the brain and spinal cord of Xt\Xt mice lead inexorably
to skeletal malformations. The abnormal skull is moulded upon the abnormal
brain. The cervical vertebrae fail to enclose the enlarged neural tissue and the
same situation, albeit to a lesser extent, is found in the sacral region. The fusions
between adjacent vertebrae in the cervical region are clearly secondary and are
limited to the processes destined to form the neural arch. There is no sign of
irregularities of the vertebral bodies (cf. talpid3, Ede & Kelly, 1964 a, b).
3. Belly spots
The data presented in Table 6 leave no doubt as to the association between Xt
and belly spotting. Similar effects of a so-called 'major' gene upon a spotting
gene have been previously described by Griineberg (1948).
Griineberg found that when mice heterozygous for microphthalmia (mi) were
crossed with his grey-lethal stock + \mi individuals showed head dots and belly
spotting absent from + / + litter-mates. Griineberg was able to show that ml
in this situation is acting as a specific genetic modifier of spotting genes.
A similar situation has recently arisen in our laboratory (G. M.Truslove,
personal communication). In syndactylism (sm, Griineberg, 1956) a head dot,
confined to animals of the sm/sm genotype, has recently arisen. This occurred
only in certain families of the stock and has now died out.
It therefore seems most probable that on the genetic background of the Xt
stock the gene acts as a genetic modifier of pre-existing spotting genes.
Extra-toes: a new mutant
577
Relationship o/Xt to other syndromes
Few syndromes known to developmental genetics provide such a range of
abnormalities as does extra-toes. The presence of a large number of concordant
abnormalities is limited to a few genes, pollex (guinea-pig), blebs (mouse),
patch (mouse) and the talpids (chicken).
The similarity to pollex is striking. Most of the features described by Scott
(1937,1938) have parallels in the mouse. The youngest monsters described by him
are aged 18£ days which corresponds to the 9^-day stage in the mouse. The
characteristic features of XtjXt are present in Scott's illustrations. The ace-ofspades shape of the roof of the rhombencephalon is clear, as is the displacement
of the mandibular arch and the ovality of the eye. The description of the brain
fits Xt/Xt well. In all subsequent stages there is good agreement of abnormalities.
Scott mentions the overgrowth of the pharyngeal arches only once, in conjunction with the presence of a fifth arch. It is clear that overgrowth of the mandibular arch must have been much less pronounced in Px than in Xt. Scott's
failure to note the late closure of the neural tube may be attributed to the same
cause. Cleft palate and harelip, reported in Px, have not been seen in Xt.
Table 10 . Comparison of some features of the my, Ph and Xt syndromes
CNS
defect
my
Ph
Xt
Blebs/
oedema
Polydactyly
*
Kidney
defect
Pigment
effect
—
+
+ , Present; —, unrecorded or absent; *, triphalangy of the pollex.
The reinvestigation of the X-rayed stock of Bagg & Little (blebs, my) by
Carter (1956, 1959) and the description of patch by Griineberg & Truslove
(1960) have shown that these genes have much in common with Xt. Carter's
demonstration that previously described 'sporadic' defects are in fact basically
attributable to one major gene enabled him to construct four 'pedigrees of
causes' based upon CNS abnormalities, blebs/oedema, kidney defects and
faulty morphogenesis of the limbs. Patch and extra-toes share many of these
features (Table 10). The pigment defects include spotting in patch and a shifting
of the pigment border in black-and-tan my\my mice. This may really be a hair
defect (as Carter assumes) or a secondary effect of a dorsal bleb on skin structure.
The + sign opposite extra-toes refers to belly spotting seen in Xt heterozygotes.
It is not to be assumed, however, that the mechanisms underlying my, Ph and
Xt are identical or even similar. The defects described are common, as Carter
points out, in embryos damaged at the 8^-9^ day stages by teratogens. If nothing
more, however, it seems that the lesions of my, Ph and Xt are produced by genes
acting at a common stage of development.
578
D. R. JOHNSON
The talpid group of alleles in the chicken (Cole, 1942; Abbott, Taylor &
Abplanalp, 1959, 1960; Ede & Kelly, 1964a, b) are polydactylous monsters
superficially similar to the group described above. Ede & Kelly, however,
describe mesodermal abnormalities such as irregular vertebral bodies and failure
of mesenchymal condensations, and regard the syndrome as mesodermal in
origin.
CONCLUSIONS
Most of the abnormalities of the XtjXt phenotype can be ultimately traced
back to one of three anomalies: the disturbed neural tube, overgrowth of the
pharyngeal arches and limb-buds and, later, oedema. As the first two abnormalities both become visible at the 9-day stage it is not possible to suggest a causal
relationship on temporal grounds. Three points of view are possible. First, the
overgrowth of the pharyngeal arches may mechanically affect the closure of the
neural tube. This is a possibility, as the area of non-closure is in the region of the
pharyngeal arches and no other region of the neural tube is persistently open.
On the other hand, the hind brain is one of the two regions most commonly
affected by non-closure, and many cases of hind-brain dysraphism not associated
with spina bifida are known. Secondly, there may be an abnormality of the
neural plate which delays its closure and in some way affects the pharyngeal
arches and the limb-buds. An abnormality of the CNS could easily account for
the derangements seen in axial structures. Involvement of the appendicular
skeleton is less easily explained. However, M. S. Deol (unpublished) has suggested that in some mutants the abnormalities of the nervous system may not be
confined to the neural tube but expand over the ganglia and nerves as well, and
when this happens in the pectoral or pelvic region the limbs may be affected in
consequence. The present paper contains no evidence to confirm or deny this
hypothesis. Thirdly, an unknown pre-existing factor may affect the development
of limb-buds, pharyngeal arches and neural tube.
If we accept that the overgrowth of the neural tube is primary rather than
secondary, then both this and the enlargement of the limb-buds and pharyngeal arches could be due to overgrowth, possibly as a result of a derangement of
the reciprocal ectodermal/mesodermal interaction present during organogenesis.
This could also explain the late proliferation of the nasal and retinal epithelia
and the appearance of extra vibrissae. Also in favour of this hypothesis is the
lack of abnormalities where ectoderm and endoderm interact without the
intervention of mesoderm; the pituitary body and the VER of the tail are examples of this. Both are normal in XtjXt mice.
The position reached by this line of reasoning is similar to that of Scott (1938)
in Px. He postulated the production in PxjPx embryos of an intracellular growth
factor 'for a short time after approximately 17i days copulation age'. Carter
(1959) in reviewing his four 'pedigrees of causes' for my remarked that no causal
connexions between them had been traced, but goes on to postulate that con-
Extra-toes: a new mutant
579
nexions may exist because of the significant statistical associations between parts
of the syndrome. Griineberg & Truslove (1960) are inclined to ascribe the
defects of PhjPh to hydrops which is present from the 8-day stage onwards.
If we accept the concept of unitary primary gene action we also accept that
the parts of extensive syndromes such as those produced by the genes my, Ph, Px
and Xt are related. In the absence of factual evidence to support this hypothesis,
however, the investigator is liable to fall between two stools: either to admit
defeat (as did Carter) or to propose a blanket explanation (as have Scott and
myself) which runs the risk of being so general as to explain nothing. It is hoped,
however, that further studies of Xt mice at present contemplated will allow a more
precise explanation to be offered at a later date.
SUMMARY
1. Extra-toes (Xt) is a new semi-dominant gene in the mouse. XtjXt animals
die in utero or at birth. They have multiple abnormalities including paddleshaped feet with up to eight or nine digits, hemimelia, disturbed spinal cord and
brain, nose, eye and ear, and oedema. The heterozygote has pre- and postaxial polydactylism of all four feet.
2. The Extra-toes homozygote can first be recognized as a 9-day embryo by
excessively large pharyngeal arches and persistently open neural tube.
3. Many abnormalities of the brain and sense organs can be traced back to
malformation of the neural tube.
4. Malformations of the upper jaw region and limbs can be traced back to
overgrowth from the 9-day stage onwards.
5. A third series of malformations due to oedema (first seen at 13 days) is
described.
6. The possible interrelations of these three groups of abnormalities to each
other, and the relationship of the Xt syndrome to other gene effects in the mouse,
chicken and guinea-pig are discussed.
RESUME
Extra-toes: un nouveau gene mutant provoquant de
nombreuses anomalies chez la Souris
1. Extra-toes (Xt): nouveau gene semi-dominant chez la Souris. Les
animaux Xt/Xt meurent dans l'uterus ou a la naissance. Us presentent de
nombreuses anomalies parmi lesquelles des pieds en forme de palette avec plus
de 8 ou 9 orteils, de l'hemimelie, l'alteration de la moelle epiniere et du cerveau,
du nez, de l'oeil, de l'oreille et de l'cedeme. Les quatre pieds des sujets heterozygotes presentent de la polydactylie pre et postaxiale.
2. On recommit le type homozygote de la mutation extra-toes chez l'embryon
de 9 jours par la presence d'arcs branchiaux tres larges et d'un tube neural
ouvert.
580
D.R.JOHNSON
3. De nombreuses anomalies du cerveau et des organes des sens peuvent
etre dues aux malformations du tube neural.
4. Les malformations de la region de la machoire superieure et des membres
pourraient provenir d'excroissances apparaissant apres le 9eme jour.
5. Description d'une troisieme serie de malformations due a un cedeme
(premiere observation a 13 jours).
6. La discussion porte sur les relations possibles entre les trois groupes
d'anomalies et la relation entre le syndrome Xt et d'autres effets du gene chez
la souris, le poulet et le cobaye.
I am indebted to Professor H. Griineberg, F.R.S., for his constant encouragement and
criticism, and to Drs Gillian M. Truslove and M. S. Deol for much useful discussion and
advice. I am much indebted to Mr A. J. Lee who prepared most of the Text-figures. The work
was supported by a grant from the Medical Research Council which is gratefully acknowledged.
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{Manuscript received 23 January 1967)