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/. Embryol. exp. Morph. Vol. 21, 2, pp. 347-60, April 1969
347
Printed in Great Britain
Development of luxoid (lu) skeletal defects in vitro
By D. J. BURDA 1 & E. M. CENTER 1
From the Department of Anatomy, Stanford University School of Medicine
Skeletal abnormalities in the hind limb of the homozygous luxoid mutant
mouse (lujlu) have been described by Green (1955) and Forsthoefel (1958, 1959).
These defects include a reduction in the size of the tibia, a slight thickening of
the fibula and a failure to form normal condyles on the distal portion of the
femur. Chondrogenesis and ossification are also slightly retarded in these limbs.
The early work of Murray & Huxley (1925) and of Fell and co-workers (1929,
1934) indicated that isolated skeletal elements are self-differentiating in vitro;
however, there has been no experimental evidence to indicate whether luxoid
skeletal defects likewise are self-differentiating, or whether these features are
dependent upon or the result of continuous interactions of the embryonic
skeletal elements with one another or with nearby tissues. The purpose of the
present study thus has been to determine the effects of disarticulation and
isolation on the manifestation of the luxoid characteristics in vitro.
The luxoid mutant used in this study was discovered in the C57BL/6Sfd strain
in the animal colony of the Anatomy Department of the Stanford University
School of Medicine. In a study by Center, Hunter & Dodge (1967) the mutant
was shown to be morphologically and genetically identical with the luxoid
mutant first described by Green (1955). In the present investigation comparisons
were made between the chronological changes in the gross and histological
features of normal and luxoid skeletal elements as isolates. This work extends
the embryological observations of Forsthoefel (1959) on the intact limb of the
luxoid mutant mouse.
MATERIALS AND METHODS
Twenty-four litters were obtained from matings between luxoid heterozygotes. The size of the litters ranged from three to nine embryos; the over-all
ratio of normal to luxoid embryos found in these litters was approximately 2:1.
In the C57BL/6Sfd line of luxoid embryos used in this study the following
criteria distinguish the luxoid embryo from the normal one. In situ, the hind limb
of the luxoid embryo is somewhat shorter and thinner, the footplate shows
excessive preaxial growth (in many cases on all four limbs) and the hind feet are
flexed medially. After excision, the reduced tibial blastema of the luxoid embryo
is visible with transmitted light.
1
Authors' address: Department of Anatomy, Stanford University School of Medicine,
Stanford, California 94305, U.S.A.
23
JEEM2I
348
D. J. BURDA & E. M. CENTER
The developmental age of the embryos was determined according to the
external morphological characteristics outlined by Griineberg (1943). The
developmental age usually correlated well with vaginal plug timings, in which
day 'zero' designated the day on which the copulation plug was observed.
A total of 211 skeletal elements was removed from the hind limbs of normal
and homozygous luxoid mouse litter-mates ranging in age from 13 days through
15 days of gestation (Table 1). The cartilaginous anlagen of femora, tibiae and
Table 1. Number of skeletal elements cultured from
normal and luxoid mouse embryos
14--141
13-131
15
A
CAM
14
14
12
2
2
1
2
1
2
0
0
10
18
18
17
105
cu
CAM
CU
CAM
Total
7
6
7
7
2
7
5
2
8
2
1
2
2
2
2
1
0
1
1
0
1
32
25
29
35
29
37
3
3
44
0
0
31
3
3
17
0
0
4
12
12
211
ON ON ON
Luxoid Fe
Fi
T
Normal Fe
Fi
T
Articulated
Luxoid
Normal
Total
CU
ON ON
Disarticulated
CU, cultured on grid; CAM, chorioallantoic explantation; Fe, femur; Fi, fibula, T, tibia.
fibulae were stripped of mesenchyme by means of tungsten needles and were
cultured either in the articulated or disarticulated state, using the grid method or
chorioallantoic explantation. Contralateral hind limb anlagen were removed
and fixed at the time of culture so as to provide non-cultured control specimens.
For the grid method the elements were placed on the surface of ultrathin
Millipore filters with a thickness of 25/6 and a pore size of 0-45/t. The filters
were supported on stainless steel grids which were placed over the center wells
of standard organ culture dishes. The culture medium consisted of a mixture of
80 % Waymouth's medium, 10 % horse serum and 10 % chick embryo extract,
supplemented with penicillin (100 units/ml), streptomycin (100 /tg/ml) and
Fungizone (2 ^g/ml). The cultures were gassed with 5 % CO2 m an " a n d maintained for 1-5 days at 38 °C.
For chorioallantoic explantation, apertures were made in the shells of white
leghorn hens' eggs maintained to 9 days of incubation. At this time the mouse
skeletal rudiments were placed on the chorioallantoic membranes (CAM) of the
host chick embryos. Excess fluid was withdrawn from the vicinity of the elements,
the apertures in the shells were sealed with Cellophane tape and the eggs were
returned to incubation at 38 °C for 1-5 days.
Luxoid skeletal defects
349
For histological study, cultured and non-cultured rudiments were fixed in
95% alcohol, embedded in Paraplast, sectioned at a thickness of 10/* and
stained with Hematoxylin and Eosin. The sections were examined at magnifications ranging from 35 to 430 x .
Table 2. In vitro increase in length of disarticulated skeletal elements
from normal and luxoid mouse embryos
Specimen no.
Type of element
CU18
CU36
CU43
CU30
CU46
CU18
CU36
CU43
CU30
CU46
CU18
CU43
CU30
CU46
CU56
CU57
CU56
CU57
CU56
CU57
CU13
CU10
CU13
CU10
CU47
CU10
CU49
CU41
CU40
CU41
CU40
CU41
CU40
CAM 65
CAM 67
CAM 92
CAM 51
CAM 93
CAM 55
Normal femur
Normal femur
Normal femur
Luxoid femur
Luxoid femur
Normal tibia
Normal tibia
Normal tibia
Luxoid tibia
Luxoid tibia
Normal fibula
Normal fibula
Luxoid fibula
Luxoid fibula
Normal femur
Luxoid femur
Normal tibia
Luxoid tibia
Normal fibula
Luxoid fibula
Normal femur
Luxoid femur
Normal tibia
Luxoid femur
Normal fibula
Luxoid fibula
Luxoid fibula
Normal femur
Luxoid femur
Normal tibia
Luxoid tibia
Normal fibula
Luxoid fibula
Luxoid femur
Luxoid tibia
Normal femur
Luxoid femur
Normal tibia
Luxoid tibia
Increase
Age at
in length
culture
after 2 days
(days'
gestation)
(%)
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13*
13*
13*
m
13*
13*
14
14
14
14
14
14
14
15
15
15
15
15
15
13
13
14
14*
14|
14*
450
190
10-5
15-3
381
31-6
250
26-7
111
22-2
500
500
33-3
33-3
200
167
200
6-7
200
22-5
171
190
200
250
250
190
250
22-2
250
350
200
70
33-3
45-5
500
20-5
200
400
54-7
CU, grid method specimens; CAM, chorioallantoic method specimens.
23-2
350
D. J. BURDA & E. M. CENTER
RESULTS
The results obtained from the two different culture methods will be discussed
separately. Only those features which serve to distinguish normal from luxoid
elements will be described. For both methods the limbs are grouped chronologically according to developmental age (Griineberg, 1943).
Of the 211 elements which were cultured, the percentage increase in length
was determined for 39 disarticulated elements which were selected for comparison after 2 days in culture at each stage. These data are presented in
Table 2. Although it is readily apparent that growth does occur for each element, comparisons could not be made between the growth capacity of normal
and luxoid elements because of insufficient numbers for statistical analyses.
Grid culture method
13- and 13\-day limbs. At the time of removal the skeletal anlagen consist of a
cartilaginous matrix surrounded by dense mesenchyme. Although it is difficult to
distinguish luxoid from normal elements, thickened areas on the distal end of the
normal femur suggest incipient condyle formation (Fig. 1 A, B). In histological
sections of the normal and luxoid femora the majority of chondrocytes appear
to represent a morphologically homogeneous population, except for a limited
central area in the femora where hypertrophy has begun (Fig. 1C). In this
region the chondrocytes exhibit swollen nuclei, and the cytoplasm is vacuolated.
The zone of hypertrophy is larger in the normal femur than that in the luxoid
element. In the normal femur a well-developed layer of basophilic osteoblasts
can be seen along the inner border of the perichondrium surrounding the hypertrophied zone. The perichondrium of the luxoid femur shows sparsely scattered
osteoblasts at this stage.
When the elements are cultured in the articulated state normal and luxoid
features become easily distinguishable after 3 days. The normal limb shows
well-developed condyles on the femur, a typical normal tibia with a malleolus
and a slender fibula (Fig. ID). In contrast, the luxoid femur lacks normal
condyles, the tibia is short and slender and the fibula is thickened (Fig. 1E).
When the 13-day normal and luxoid femora are disarticulated and maintained
in culture for 2 days there is a dense outgrowth of fibroblasts. The distal end of
the normal femur begins to develop a shallow intercondylar fossa, which is
lacking in the luxoid femur (Fig. 2 A, B). When these are maintained beyond
2 days typical condyles develop in each of the normal femora but fail to appear
on the luxoid elements. The proximal end of each normal and luxoid element
develops a head and trochanters. Histologically, both the normal and luxoid
cartilaginous anlagen show an extensive zone of hypertrophy in the center of
the diaphysis. Peripheral to this is a zone of proliferation, with irregularly
arranged chondrocytes. At the epiphyses are zones of reserve cartilage (Fig. 2C).
Disarticulated tibiae cultured for 2 days develop features consistent with those
Luxoid skeletal defects
D
FIG. 1
A. Non-cultured 13-day normal femur, x 36.
B. Non-cultured 13-day luxoid femur. x36.
C. Longitudinal section of non-cultured 13-day luxoid femur. Note beginning of
hypertrophied zone in center of diaphysis. x 100.
D. 13-day normal femur, tibia and fibula cultured in the articulated state for
3 days. Arrows indicate condyles on femur, x 30.
E. 13-day luxoid femur, tibia and fibula cultured in the articulated state for
3 days. Arrow indicates absence of condyles on femur, x 30.
351
352
D. J. BURDA & E. M. CENTER
D
FIG.
2
A. Distal portion of 13-day normal femur cultured in the disarticulated state for
2 days, x 32.
B. Distal portion of 13-day luxoid femur cultured in the disarticulated state for
2 days, x 32.
C. Longitudinal section of 13-day luxoid femur cultured for 2 days. Note zones
of hypertrophy, proliferation and reserve cartilage, x 65.
D. 14-day normal femur cultured in the disarticulated state for 2 days, x 30.
E. 14-day luxoid femur cultured in the disarticulated state for 2 days, x 30.
F. 14-day normal tibia cultured in the disarticulated state for 2 days. Note
marrow cavity in center of diaphysis. x 30.
G. 14-day luxoid tibia cultured in the disarticulated state for 2 days. Note lack of
marrow cavity, x 30.
Luxoid skeletal defects
353
observed in vivo. The distal portion of the normal tibia begins to develop a
malleolus, which is lacking in the luxoid tibia. The luxoid fibula becomes
thickened, in contrast to the slender normal fibula.
14- and 14\-day limbs. When skeletal rudiments are removed at 14 days'
gestation the proximal end of each normal and luxoid femur contains a head
and trochanters. Distally, condyles are barely distinguishable on the normal
femur, but are absent from the luxoid femur. Histologically, the 14-day normal
and luxoid femora show a large central zone of hypertrophy and uniform rows
of small flattened cells which constitute the zone of proliferation. Whereas
numerous basophilic osteoblasts occur along the inner layer of the normal
periosteum, osteoblasts are relatively sparse in the luxoid periosteum. Also,
a faint bone collar can be identified in the normal femora, but is lacking in the
luxoid rudiments. Osteoblastic activity in the normal tibia and fibula lags behind
that in the normal femur. The luxoid counterparts of these elements likewise
show retardation.
After 2 or more days in culture in the articulated state, typical normal and
luxoid features develop, respectively, for each element. The disarticulated
anlagen maintained 2 days in culture likewise show differences from one another
(Fig. 2D, E). Where prominent condyles develop on the normal femur the distal
end of the luxoid femur develops a knob-like expansion which lacks an intercondylar fossa. The normal tibia shows slight condyle formation and tapers
distally to form a malleolus. In contrast, the luxoid tibia is retarded and lacks
any distal process. After 2 or more days in vitro the luxoid fibula is thickened,
whereas the normal fibula becomes attenuated.
The normal and luxoid elements also differ with respect to the formation of
a marrow cavity. In the cultured normal tibiae, cavitation can be distinguished
in the diaphyses of gross and histological specimens (Fig. 2F). The luxoid
elements lack any marrow cavity at this stage, although zones of hypertrophy
and proliferation are evident (Fig. 2G). Normal and luxoid elements show
numerous basophilic osteoblasts along the inner layer of the periosteum.
A periosteal bone collar, however, is still absent in the luxoid rudiments. In the
normal femora the bone collar, which was barely present at the time of culture,
does not show any obvious increase in size in vitro.
15-day limbs. At the time of explantation the skeletal anlagen of the hind limb
are well developed. The normal femur exhibits prominent condyles distally, as
well as a large and distinct marrow cavity in the diaphysis. Although the distal
end of the luxoid femur is expanded, an intercondylar fossa is lacking. Marrow
cavities are beginning to form in the luxoid anlagen at this stage. After 2 or
more days in culture in the articulated state the elements continue to show
features consistent with their genotype (Fig. 3 A, B).
When disarticulated elements are maintained in vitro for 2 days the luxoid
rudiments lag behind the normal ones with respect to the size of the marrow
cavity. The luxoid tibia, in particular, shows only a narrow band indicating
354
D. J. BURDA & E. M. CENTER
Luxoid skeletal defects
355
slight cavitation (Fig. 3C). In the normal and luxoid specimens a bone collar
can be detected beneath the osteogenic layer of the periosteum. Distinct zones
of hypertrophy and proliferation are also easily identified.
Chorioallantoic explanation
Because of the technical difficulties involved in explantation onto the chorioallantoic membrane (CAM) of the chick, fewer elements were obtained than
was the case with the grid method. A total of 45 disarticulated skeletal rudiments
was successfully maintained on the CAM. Within 24 h after explantation these
elements were penetrated by blood vessels from the host. In each instance the
periosteum was invaded by numerous small vessels, after which a larger vessel
would penetrate the diaphysis as a periosteal bud.
In all age groups gross features characteristic of normal and luxoid elements
manifest themselves after two or more days on the CAM. In the normal femur
the distal portion becomes expanded and shows well-defined condyles, whereas
the distal portion of the luxoid femur merely develops a club-shaped expansion
(Fig. 3D, E). The tibiae likewise develop according to their genotype. The
normal tibia shows an enlarged epiphysis proximally and a tapered malleolus
distally (Fig. 3F). Despite an advantageous, well-vascularized position on the
CAM, the luxoid tibia develops as a slender, rudimentary element (Fig. 3G).
Histologically, 13-day normal and luxoid femora cultured for 2 days show
well-defined zones of hypertrophy, proliferation and reserve cartilage, as well as
the beginning of a periosteal bone collar (Fig. 3H). When these elements are
maintained beyond 2 days on the membrane, the bone collar increases in size,
calcification begins to occur in the central zone of cartilage and a marrow cavity
develops; however, the degree of calcification and cavitation in the luxoid
elements is retarded.
FIG. 3
A. 15-day normal femur cultured in the articulated state for 2 days. Well-developed
condyles are present, x 30.
B. 15-day luxoid femur cultured in the articulated state for 2 days. Normal condyles
are lacking, x 30.
C. 15-day luxoid tibia cultured in the disarticulated state for 2 days, x 30.
D. 14-day normal femur cultured in the disarticulated state on the CAM for 2 days.
x30.
E. 14-day luxoid femur cultured in the disarticulated state on the CAM for 2 days.
x30.
F. 14-day normal tibia cultured in the disarticulated state on the CAM for 2 days.
x30.
G. 14-day luxoid tibia cultured in the disarticulated state on the CAM for 2 days.
x30.
H. Longitudinal section of 13-day luxoid femur cultured in the disarticulated state
on the CAM for 2 days, x 90.
356
D. J. BURDA & E. M. CENTER
After 2 days on the CAM histodifferentiation of the 14-day rudiments is
excellent. In the normal femur the greater portion of the diaphysis consists of
greatly hypertrophied chondrocytes, although calcification is not as yet present.
Peripheral to these are wide bands of proliferation. Osteoblasts now form a
continuous layer along the periosteum, beneath which is the bone collar. The
luxoid femur exhibits a large central zone of hypertrophy, peripheral zones of
proliferation, numerous osteoblasts and a slight bone collar. As development
proceeds beyond 2 days on the CAM normal and luxoid elements continue to
differentiate, and a marrow cavity develops in each rudiment. In the luxoid
rudiments, however, the extent of ossification always lags behind that seen in
the normal ones.
DISCUSSION
The results of this study indicate that skeletal elements isolated from the hind
limb of the luxoid mouse embryo at 13 or more days' gestation have the capacity
for self-differentiation in vitro. At 13- to 131-day s of gestation chondrification of
the luxoid tibia is considerably retarded in vivo. The luxoid femur and fibula are
retarded to a lesser degree, although they lag behind their normal counterparts
with respect to the establishment of the various cartilaginous zones, as indicated
histologically by the smaller central areas of hypertrophy seen in the luxoid
elements. When these are disarticulated, virtually stripped of surrounding
mesenchyme and cultured for 2 or more days, they continue to exhibit the lag
in chondrogenesis which occurs in vivo. This retardation cannot thus be attributed to factors in the intact limb which might suppress normal growth and
differentiation of the skeletal elements.
In luxoid limb rudiments the formation of a bone collar also lags behind that
seen in the normal condition. The bone collar can be identified in normal 14to 14|-day femora but is lacking in their luxoid counterparts. With the grid
method of culture used in the present study the bone collar is maintained in
normal elements, but it does not show any increase in size. If the collar is not
present at the time of explanation it fails to develop in culture.
With the CAM method, however, the collar develops in both normal and
luxoid elements, including those which lack any indication of a collar at the
time of explantation. Development of a marrow cavity appears to be dependent
upon the presence of vascular elements, since well-developed cavities become
apparent regardless of whether or not cavitation has begun at the time the
rudiments are explanted. The luxoid elements, however, show retardation in the
extent of cavitation in comparison with normal elements of the same age. In
contrast to the CAM technique, the grid method of culture does not seem to be
conducive to the establishment of a marrow cavity. Chick rudiments cultured by
the watchglass method likewise fail to develop marrow cavities (Fell & Robison,
1929).
Our results agree with the observation that cartilage cells must first hyper-
Luxoid skeletal defects
357
trophy before the perichondrium can become osteogenic (Fell, 1932). The lag in
hypertrophy illustrated by the luxoid rudiments was always followed by a lag in
osteogenic activity in the perichondrium.
According to Forsthoefel (1959), ossification of luxoid elements reaches a
state comparable to that of their normal counterparts at the time of birth. This
was not tested with the grid method of culture because of hydration artifacts
and gross abnormalities which commonly occur in mouse skeletal elements
maintained beyond 4 or 5 days in vitro (Biggers, Gwatkin & Heyner, 1961).
Although ossification takes place more readily in CAM grafts, insufficient
numbers of luxoid and normal elements were obtained from prolonged periods
in culture to verify Forsthoefel's observation under these conditions.
The morphological integrity of the skeletal elements is maintained equally well
with the two culture techniques used in the present study. Of importance is the
nature of the luxoid defect which involves a failure to form normal condyles on
the distal portion of the femur. This defect occurs also in Strong's luxoid mutant
(Forsthoefel, 1962) and to a lesser degree in the luxate mutant (Carter, 1951).
In the latter there is a slight reduction in the size of the condyles and in the
depth of the intercondylar fossa. Interaction of Strong's luxoid gene with Green's
luxoid and Carter's luxate likewise results in abnormal development of condyles
(Forsthoefel, 1962). The presence of tibial hemimelia in all of these mutants has
been suggested as a factor which might exert some inhibiting influence on the
morphogenesis of the distal portion of the femur. The results of our study show,
however, that when the luxoid femur of 13 or more days' gestation is cultured as
an isolate, normal condyles fail to form. This defect is thus not dependent upon
the continuous association of femur with tibia or with other tissues of the hind
limb, at least in the stage of development during which the defect becomes
morphologically distinct. Attention should thus be focused on interactions
which may occur prior to this period.
Efforts to culture disarticulated elements of early developmental stages have
not met with much success because of technical difficulties (Griineberg, 1963).
We have obtained a few 12-day rudiments which appear to be self-differentiating
in the articulated state in vitro, but isolation of early skeletal 'elements' is
precluded by the continuous mesenchymal condensation which represents
femur, tibia and fibula, and possibly some skeletal muscle as well (Forsthoefel,
1963).
The sequence of mesenchymal condensations may be an important factor in
the induction of tibial hemimelia, accompanied by a thickening of the fibula.
Since the blastemal condensation apparently begins postaxially, an overabundant
accumulation of postaxial mesenchyme might result in an enlarged fibula with
a correspondingly reduced tibia (Forsthoefel, 1963). Our results have shown
that this predominance of the fibula is maintained in vitro, as indicated by the
relatively thicker diaphysis of the luxoid fibula in comparison with that of the
luxoid tibia cultured for 2 or more days.
358
D. J. BURDA & E. M. CENTER
Our results also indicate that the autonomous nature of the luxoid defect in the
mouse is similar to that observed for various skeletal anomalies found in the
chick embryo. Included among the latter are the talpid3 mutant (Hinchliffe &
Ede, 1968), the Creeper mutant (Hamburger, 1941; Rudnick, 1945; Elmer,
1968) and diplopod (Abbott & Kieny, 1961).
The capacity for a luxoid element to elongate in vitro does not appear to be
affected by its genotype. As indicated in Table 2, a wide range of growth was
exhibited by normal and luxoid elements maintained in culture for 2 days.
Because of insufficient numbers, however, statistical analyses were not applied
to these data. Moreover, no conclusions could be drawn concerning the capacity
for over-all growth, since no measurements were taken of wet weight or dry
weight.
Morphological differentiation of a skeletal element is thought to take place
during the procartilage and early cartilage phase of development (Fell, 1956).
During this time the element does not seem to be subject to extrinsic factors, as
indicated by the excellent morphogenesis of the rudiments in the grid culture
environment and on the CAM. Bateman (1954) suggests that there is an almost
total lack of extrinsic control on the pattern of development of individual mouse
bones throughout gestation. Once a bone begins to ossify, however, it seems to
become dependent upon the presence and availability of vascular elements, since
we found that ossification proceeded adequately only on the highly vascular
chorioallantoic membrane.
SUMMARY
1. Skeletal elements isolated from the hind limbs of luxoid (lu) and normal
mouse embryos of 13-15 days' gestation maintain their morphological integrity
in vitro.
2. Articulated and disarticulated anlagen of femora, tibiae and fibulae
maintain typically luxoid features when cultured with the grid method or as
explants on the chorioallantoic membrane (CAM). The gross and histological
nature of the limb defects in the luxoid embryo are not dependent upon continuous association of the femur, tibia and fibula during the stages examined in
the present study.
3. Chondrification is obtained with both culture techniques; however,
ossification and the establishment of a marrow cavity are obtained only with
the CAM method.
RESUME
Developpement in vitro de deficiences squeiettiques du type 'luxoide' (lu)
1. Des elements squelettiques isoles de membres posterieurs d'embryons de
souris normales et 'luxoides' (lu), du 13 au 15e jour de la gestation, conservent
leur integrite morphologique in vitro.
2. Des ebauches articulees et desarticulees de femurs, de tibias et de perones
Luxoid skeletal defects
359
conservent des caracteres typiquement 'luxoides' quand on les cultive selon la
methode de la grille ou en explants sur la membrane chorioallantoidienne
(MCA). Les caracteres generaux et histologiques des deficiences des membres de
l'embryon 'luxoide' ne dependent pas d'une association continue du femur, du
tibia et du perone pendant les stades examines dans ces recherches.
3. On obtient la chondrification avec les deux methodes de culture; neanmoins, 1'ossification et la formation d'une cavite medullaire ne s'obtiennent
qu'avec la methode MCA.
We would like to express our appreciation to Mrs Alice Dodge and Miss Laurie Paavola
for technical assistance, and to Mr Max Millsap for the photography. This work was supported
by research grants no. HD01312-06 and HD01679-02 from the National Institute of Child
Health and Human Development, United States Public Health Service.
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