/. Embryol. exp. Morph. Vol. 21, 2, pp. 285-94, April 1969
285
Printed in Great Britain
Polysyndactyly., a new mutant gene in the mouse
By D. R. JOHNSON 1
From the M.R.C. Experimental Genetics Unit,
University College London
Our understanding of the mechanisms underlying mutant genes affecting the
limb skeleton is very incomplete. At this stage description of new phenotypes is
still valuable, especially when they differ markedly from those previously described and have counterparts in man.
MATERIALS AND METHODS
The gene for polysyndactyly (Ps) arose during a neutron irradiation experiment at the M.R.C. Radiobiological Unit at Harwell (Batchelor, Phillips &
Searle, 1966), and was offered to the author in November 1964. The mice from
Harwell were outcrossed to CB $? (the Fx of CBA/Gr &?xC57 BL/Gr $?).
The stock was outcrossed again in late 1966 to improve fertility. Rigorous
brother/sister mating has not been practised.
Embryos from Ps/ + xPs/ + matings were fixed in Bouin's solution, sectioned at 7-5 or 10/* according to age and stained with H & E. In most cases
only the limbs were sectioned (left fore and hind limbs transversely, right ones
tangentially). These included groups of 2 PsjPs, 2 Psj + and 2 + / + embryos
aged 15 and 14 days and six 13-day embryos which could not be classified by
inspection. Twelve 12-day and five 11-day embryos were sectioned intact in the
transverse plane. Six litters aged 14 days were vitally stained with 1/40,000 Nile
blue sulphate in isotonic saline.
Methylene blue preparations were made following Griineberg's (1953) protocol (Table 1). Alizarin red S clearance preparations were made by a modification
of Dawson's (1926) technique (Table 2). The feet of 15 Ps\ + and 15 + / + littermates were preserved in 10 % formol saline for detailed examination.
GENETICS
Polysyndactyly is a semi-dominant gene lethal at birth when homozygous
(Table 3). In matings between heterozygotes the ratio of Psj + to + / + is a
good fit to 2:1, but there is a deficiency of Ps IPs which are stillborn or perish
1
Author's address: M.R.C. Experimental Genetics Unit, University College London,
4 Stephenson Way, London, N.W. 1, England.
19-2
286
D. R. JOHNSON
quickly. In matings of Ps I + x + / + there is a good fit to a 1:1 ratio. From the
time when they can first be classified (13-14 days of embryonic life) until birth
there is no deficiency of Ps/Ps (Table 4).
Table 1. Methylene blue preparations
Age
(days)
15
16
Ps/ +
Ps/Ps
Total
12
8
11
31
10
1
11
22
31
14
34
79
9
5
12
26
Newborn
Total
Table 2. Alizarin red S preparations
Age
(days)
+
Newborn
28
/+
Ps/+
Ps/Ps
Total
11
23
1
—
18
40
6
17
Table 3. Segregation o/Ps at birth
Mating type
+ /+
Ps/+
Ps/Ps
Unclassinable
Total
Mean litter size
l.Ps/+ xPs/ +
66
115
15
10
206
6-4
225
217
—
2
444
7-3
2.Ps/+ x +l + \
+ /+ xPsl+j
Table 4. Segregation 0/Ps in litters of embryos
Age
(days)
10-12
13
14
15
16-18
Total
Solid
moles
Total
Mean
litter
size
+/ +
Ps/ +
Ps/Ps
Unclassifiable
—
5
1.1
14
13
43
—
12
20
31
31
—
6
18
19
18
42
28
0
0
0
3
4
3
5
1
45
55
52
69
63
90
9-2
7-4
8-6
7-9
94
61
70
16
284
8-6
Linkage data (Table 5) was kindly supplied by Dr A. G. Searle (M.R.C.
Radiobiological Unit, Harwell). Ps was found to be on Linkage Group YIII.
Males and females of the constitution +Ps+ + /m+Ptb were backcrossed to
misty browns. Classification for pintail was difficult at times because of poor
Polysyndactyly
287
expression, therefore only the results for Ps, m and b are regarded as fully reliable
and given here. Ps is presumed to lie between m and b. The recombination
frequencies in the two sexes do not differ significantly and the combined results
yield the following estimates of crossover distances:
m ...0-6 ...Ps ... 8-2 ...b.
Table 5. Results of + Ps + /m + b x m + b/m + b crosses
Heterozygous
parent
,
+Ps +
Numbers with phenotypes
m+b
+Psb
mPs +
Male
Female
Total
53
114
167
44
101
145
4
13
17
1
1
2
Total
0
0
0
105
237
342
11
Recombination frequencies between Ps and 6 = 6-7 + 2-4 % (£<?) and 90 ± 1-9 % ($9).
Recombination frequencies between Ps and m = 1-0 ±0-9 % (<$<$) and 0-4 ±0-4 % ($$).
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Age (days)
Text-fig. 1. Representative growth curves of 3 Ps/+ mice and
their 5 + / + litter-mates.
THE ADULT
Psj + mice are smaller than their sibs at birth (44 normals from nine litters
had a mean birthweight of 1-533 g.: their 31 Ps/+ sibs weighed 1-496 g.
t = 6-22, P < 001). By 20 days they are about 10 % lighter (Fig. 1).
In fore and hind feet the claws are reduced and nail-like (Fig. 2). Psj + mice
are active at ground level but do not climb well. They are unable to hang inverted from the wire mesh top of their cage, a feat easily performed by their
Text-fig. 2. Ventral views of adult right hind feet (A-C) and left forefeet (D, E) from the/\y stock. Each foot is drawn first intact,
then as a clearance preparation. A, A', + / + . B, B',Ps/ + . C, C',Ps/+. D, D', +/ + . E, E',Ps/ + .
5 mm
2:
O
to
oo
Polysyndactyly
289
normal litter-mates. Soft tissue syndactylism is common in the hind foot, where
the first toe is never free, and rare in the forefoot (Table 6). There are occasional
fusions amongst the tarsalia and carpalia, the os tibiale primum being fused to
the tibiale, and carpal II to V. The terminal phalanges are poorly developed and
rod-like. The foramen transversum phalangis, if represented, is seldom completely surrounded by bone. In the hind foot the joint between terminal and
subterminal phalanges is abnormal: in many cases there is a fusion and in
some the joint faces are imperfectly aligned. Twelve of the 30 hind feet examTable 6. Webbing of digits in the feet of 14 adult Ps/ + mice
Digits webbed
I-II
II-III
III-IV
IV-V
No. of feet
affected
+
+
+
+
+
.
+
—
+
+
+
—
-
+
+
+
—
-
3
14
1
2
8
26
2
A
Hind feet
Forefeet
ined had a pronounced swelling under the terminal pad of digit IV (Fig. 2C).
This contained one or two small osseous elements. Small postaxial accessory
digits are rare in the hind foot (3/30) but common (19/30) in the forefoot. In
two forefeet the extra postaxial element was bifurcated.
The remainder of the limb and girdle skeleton and the axial skeleton are
completely normal.
NEWBORN
Psj + feet (Fig. 3B, E) are syndactylous at birth. An extra digit is interpolated between digits III and IV, bearing 2, 3 or more peripheral nodules, perhaps rudimentary claws. The first digit is short and broad, sometimes clearly
duplicated.
Ps/Ps feet (Fig. 3 C, F) end distally in a broad pad extending pre- and postaxially beyond the edge of the foot. Fore and hind feet are oedematous, the
oedema not extending beyond the knee or elbow joint.
EMBRYOLOGY
14 days
Most of the features of the Ps genotype can be distinguished at 14 days. PsjPs
embryos have subcutaneous oedema of the limbs and trunk. The footplates are
not divided into digits. The margin of the footplate has a ragged appearance in
tangential sections due to numerous irregular hillocks of mesoderm capped by
thickened AER (Plate 1). In the forefoot one central outgrowth dominates,
290
D. R. JOHNSON
2 mm
Text-fig. 3. Feet of newborn mice from the Ps stock. A-C left hind feet dorsal view:
D-F left forefeet ventral view. A, +/+.B,Ps/+.C, Ps/Ps. D, + / + . E, Ps/ +. F,
Ps/Ps.
becoming relatively enormous. Beneath the AER is a continuous darkly
staining band of mesoderm. Chondrification is poor and up to six metatarsals
can be seen irregularly disposed in the hind foot so that III and IV have a large
included angle.
In the heterozygote separation of the footplate into digits has begun. Between
digits III and IV, however, there are two indentations. There is preaxial enlargement of the hind foot.
The interdigital areas of cell death are disrupted in Psj + embryos, and cell
death is almost totally absent in PsjPs, both interdigitally and in the AER
(Fig. 4).
Polysyndactyly
291
Plate 1. Tangential sections through the feet of Ps mice and normal litter-mates. A, +/ + .
B, Psj +. C, Ps/Ps left forefeet 14 d; x 32. D, + / +. E, Psj +. F, Ps/Ps left hind feet 14 d; x 32.
G, Ps/Ps left hind foot 15d; x 32 (extra phalanges arrowed). H, +/ + . I, Ps/Ps right hind
feet 13 d; x40.
292
D. R. JOHNSON
13 days
PsjPs mice can first be classified on the thirteenth day. They lack the division
of the footplate into digits normally seen at this stage: the AER is wavy in the
middle or throughout its length. In transverse sections the AER is hypertrophied.
The newly appearing mesodermal condensations are irregularly placed (Plate 1,
fig. H, I). The marginal blood sinus which runs beneath the AER is enlarged
and is in close communication with it.
1 mm
Text-fig. 4. Left forefeet of 14-day-old embryos from the Ps stock vitally stained to
show cell death. A, +/ + . B, Ps/+. C, Ps/Ps.
DISCUSSION
Abnormalities resulting in the rather bizarre Ps phenotype can only be
traced back to day 13 by conventional means. At this stage we are presented
with a combination of abnormalities of AER and mesodermal condensations.
Milaire (1967) has demonstrated that in normal mid-13-day limb buds the
high RNA content and enzymic activity previously distributed along the length
of the AER become localized in those portions of the AER which overlie a
digit and decrease in interdigital portions. It is suggested that the lack of interdigital indentations in Ps is due to a failure of the activity of the AER to become
distributed into digital zones. This is borne out by the uniform appearance of
the darkly staining mesoderm below the ridge and the numerous small outgrowths each tipped with a small piece of (presumably) active AER.
Milaire also suggests that the formation of digital mesoderm depends on
ectodermal influence exerted at the tip of each' digital bud' by the corresponding
portion of the marginal ectoderm. This perhaps accounts for the induction of a
supernumerary digital bud in the abnormally large space between digits III
and IV, where the activity of the AER should be minimal.
Such a defect of the AER would account for the poor terminal differentiation
Polysyndactyly
293
of the Ps foot, the inclusion of an extra digit and polydactyly. The oedema may
be based on poor circulation caused by the failure of the blood system of the
limb to differentiate.
The abnormal placing of the skeletal blastemata is probably not a consequence
of the abnormal AER. Its occurrence suggests that perhaps a mesodermal defect
underlies the abnormality of the AER. The absence of cell death in the footplate allies Ps to talpid3 (Hinchliffe & Ede, 1967). However, in talpid3 cell
death occurs in the AER; this is not so in Ps.
This insertion of a supernumerary terminal phalanx within the footplate is
unique in the mouse. However, Nelson (1963) describes the foot of a 65-yearold woman with two extra toes inserted between the hallux and the second digit.
These had the normal number of phalanges but shared a common metatarsal.
Nelson quotes one other case of a similar nature, a 3-day-old girl reported by
Joachimsthal (1900) with six toes on each foot, the extra one being inserted
between digits IV and V.
SUMMARY
1. Polysyndactyly (Ps) is a new semi-dominant gene on linkage group VIII
of the mouse.
2. Heterozygotes and homozygotes have abnormalities of all four feet
including polydactyly and syndactyly. Homozygotes die at birth.
3. The condition can first be recognized in 13-day-old embryos, where there
are abnormalities of the AER and mesodermal condensations.
RESUME
Polysyndactyly, un nouveau gene mutant chez la Souris
1. Polysyndactyly (Ps) est un nouveau gene semi-dominant du groupe de
liaison n° VIII chez la Souris.
2. Les heterozygotes et les homozygotes presentent des anomalies des quatre
pattes, avec polydactylie et syndactylie. Les homozygotes meurent a la naissance.
3. Cet etat peut se reconnaitre initialement sur des embryons de 13 jours, ou
existent des anomalies de la cape apicale et des condensations mesodermiques.
REFERENCES
A. L., PHILLIPS, R. J. S. & SEARLE, A. G. (1966). A comparison of the mutagenic
effectiveness of chronic neutron- and y-irradiation of mouse spermatogonia. Mutation
Res. 3, 218-29.
DAWSON, A. B. (1926). A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technol. 1, 123-4.
GRUNEBERG, H. (1953). Genetical studies on the skeleton of the mouse. VII. Congenital
hydrocephalus. /. Genet. 51, 327-58.
3
HINCHLIFFE, J. R. & EDE, D. A. (1967). Limb development in the polydactylous talpid
mutant of the fowl. /. Embryol. exp. Morph. 17, 385-404.
BATCHELOR,
294
D. R. JOHNSON
. (1900). Ein weiterer Beitrag zur Lehre der Polydaktylie. Fortschr. Geb.
RontgStrahl. 4, 112-13.
MILAIRE, J. (1967). Histochemical observations on the developing foot of normal, oligosyndactylous (Os/ + ) and syndactylous (sm/sm) mouse embryos. Archs Biol. Liege, 78,
223-88.
NELSON, E. M. (1963). A report of a 7-toed foot. Anat. Rec. 147, 1-3.
JOACHIMSTHAL,
{Manuscript received 15 August 1968)