Development of luxoid (lu) skeletal defects in vitro

/ . Embryol. exp. Morph. Vol. 21, 2, pp. 347-60, April 1969 3 4 7

Printed in Great Britain

Development of luxoid (lu) skeletal defects in vitro

By D. J. BURDA1 & E. M. CENTER1

From the Department of Anatomy, Stanford University School of Medicine

Skeletal abnormalities in the hind limb of the homozygous luxoid mutantmouse (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 ofthe fibula and a failure to form normal condyles on the distal portion of thefemur. 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 luxoidskeletal defects likewise are self-differentiating, or whether these features aredependent upon or the result of continuous interactions of the embryonicskeletal elements with one another or with nearby tissues. The purpose of thepresent study thus has been to determine the effects of disarticulation andisolation on the manifestation of the luxoid characteristics in vitro.

The luxoid mutant used in this study was discovered in the C57BL/6Sfd strainin the animal colony of the Anatomy Department of the Stanford UniversitySchool of Medicine. In a study by Center, Hunter & Dodge (1967) the mutantwas shown to be morphologically and genetically identical with the luxoidmutant first described by Green (1955). In the present investigation comparisonswere made between the chronological changes in the gross and histologicalfeatures of normal and luxoid skeletal elements as isolates. This work extendsthe embryological observations of Forsthoefel (1959) on the intact limb of theluxoid mutant mouse.

MATERIALS AND METHODS

Twenty-four litters were obtained from matings between luxoid hetero-zygotes. The size of the litters ranged from three to nine embryos; the over-allratio 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 followingcriteria distinguish the luxoid embryo from the normal one. In situ, the hind limbof the luxoid embryo is somewhat shorter and thinner, the footplate showsexcessive preaxial growth (in many cases on all four limbs) and the hind feet areflexed medially. After excision, the reduced tibial blastema of the luxoid embryois 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 theexternal morphological characteristics outlined by Griineberg (1943). Thedevelopmental age usually correlated well with vaginal plug timings, in whichday 'zero' designated the day on which the copulation plug was observed.

A total of 211 skeletal elements was removed from the hind limbs of normaland homozygous luxoid mouse litter-mates ranging in age from 13 days through15 days of gestation (Table 1). The cartilaginous anlagen of femora, tibiae and

Table 1. Number of skeletal elements cultured fromnormal and luxoid mouse embryos

Disarticulated

Luxoid FeFiT

Normal FeFiT

ArticulatedLuxoidNormal

Total

CU, cultured on grid;

13-131

CU

141412

181817

ON

ON

105

CAM

221

212

00

10

14-

cuO

N

ON

O

N

767

33

44

-141

CAM

727

528

00

31

CU

212

222

33

17

15A

CAM

101

101

00

4

CAM, chorioallantoic explantation; Fe, femur; Fi,

Total

322529

352937

1212

211

fibula, T, tibia.

fibulae were stripped of mesenchyme by means of tungsten needles and werecultured either in the articulated or disarticulated state, using the grid method orchorioallantoic explantation. Contralateral hind limb anlagen were removedand 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 ultrathinMillipore filters with a thickness of 25/6 and a pore size of 0-45/t. The filterswere supported on stainless steel grids which were placed over the center wellsof standard organ culture dishes. The culture medium consisted of a mixture of80 % Waymouth's medium, 10 % horse serum and 10 % chick embryo extract,supplemented with penicillin (100 units/ml), streptomycin (100 /tg/ml) andFungizone (2 ̂ g/ml). The cultures were gassed with 5 % CO2

m an" a n d main-tained for 1-5 days at 38 °C.

For chorioallantoic explantation, apertures were made in the shells of whiteleghorn hens' eggs maintained to 9 days of incubation. At this time the mouseskeletal rudiments were placed on the chorioallantoic membranes (CAM) of thehost 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 werereturned to incubation at 38 °C for 1-5 days.

Luxoid skeletal defects 349For histological study, cultured and non-cultured rudiments were fixed in

95% alcohol, embedded in Paraplast, sectioned at a thickness of 10/* andstained with Hematoxylin and Eosin. The sections were examined at magnifi-cations ranging from 35 to 430 x .

Table 2. In vitro increase in length of disarticulated skeletal elementsfrom normal and luxoid mouse embryos

Specimen no.

CU18CU36CU43CU30CU46CU18CU36CU43CU30CU46CU18CU43CU30CU46CU56CU57CU56CU57CU56CU57CU13CU10CU13CU10CU47CU10CU49CU41CU40CU41CU40CU41CU40CAM 65CAM 67CAM 92CAM 51CAM 93CAM 55

CU, grid method

Type of element

Normal femurNormal femurNormal femurLuxoid femurLuxoid femurNormal tibiaNormal tibiaNormal tibiaLuxoid tibiaLuxoid tibiaNormal fibulaNormal fibulaLuxoid fibulaLuxoid fibulaNormal femurLuxoid femurNormal tibiaLuxoid tibiaNormal fibulaLuxoid fibulaNormal femurLuxoid femurNormal tibiaLuxoid femurNormal fibulaLuxoid fibulaLuxoid fibulaNormal femurLuxoid femurNormal tibiaLuxoid tibiaNormal fibulaLuxoid fibulaLuxoid femurLuxoid tibiaNormal femurLuxoid femurNormal tibiaLuxoid tibia

Age atculture(days'

gestation)

131313131313131313131313131313*13*13*

m13*13*1414141414141415151515151513131414*14 |14*

Increasein length

after 2 days(%)

45019010-515-338131-625026-711122-250050033-333-3200167200

6-720022-517119020025025019025022-2250350200

7 033-345-550020-520040054-7

specimens; CAM, chorioallantoic method specimens.

23-2


RESULTS

The results obtained from the two different culture methods will be discussedseparately. Only those features which serve to distinguish normal from luxoidelements will be described. For both methods the limbs are grouped chrono-logically according to developmental age (Griineberg, 1943).

Of the 211 elements which were cultured, the percentage increase in lengthwas determined for 39 disarticulated elements which were selected for com-parison after 2 days in culture at each stage. These data are presented inTable 2. Although it is readily apparent that growth does occur for each ele-ment, comparisons could not be made between the growth capacity of normaland 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 acartilaginous matrix surrounded by dense mesenchyme. Although it is difficult todistinguish luxoid from normal elements, thickened areas on the distal end of thenormal femur suggest incipient condyle formation (Fig. 1 A, B). In histologicalsections of the normal and luxoid femora the majority of chondrocytes appearto represent a morphologically homogeneous population, except for a limitedcentral area in the femora where hypertrophy has begun (Fig. 1C). In thisregion the chondrocytes exhibit swollen nuclei, and the cytoplasm is vacuolated.The zone of hypertrophy is larger in the normal femur than that in the luxoidelement. In the normal femur a well-developed layer of basophilic osteoblastscan be seen along the inner border of the perichondrium surrounding the hyper-trophied zone. The perichondrium of the luxoid femur shows sparsely scatteredosteoblasts at this stage.

When the elements are cultured in the articulated state normal and luxoidfeatures become easily distinguishable after 3 days. The normal limb showswell-developed condyles on the femur, a typical normal tibia with a malleolusand a slender fibula (Fig. ID). In contrast, the luxoid femur lacks normalcondyles, the tibia is short and slender and the fibula is thickened (Fig. 1E).

When the 13-day normal and luxoid femora are disarticulated and maintainedin culture for 2 days there is a dense outgrowth of fibroblasts. The distal end ofthe normal femur begins to develop a shallow intercondylar fossa, which islacking in the luxoid femur (Fig. 2 A, B). When these are maintained beyond2 days typical condyles develop in each of the normal femora but fail to appearon the luxoid elements. The proximal end of each normal and luxoid elementdevelops a head and trochanters. Histologically, both the normal and luxoidcartilaginous anlagen show an extensive zone of hypertrophy in the center ofthe diaphysis. Peripheral to this is a zone of proliferation, with irregularlyarranged 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 351

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 ofhypertrophied zone in center of diaphysis. x 100.D. 13-day normal femur, tibia and fibula cultured in the articulated state for3 days. Arrows indicate condyles on femur, x 30.E. 13-day luxoid femur, tibia and fibula cultured in the articulated state for3 days. Arrow indicates absence of condyles on femur, x 30.


D

FIG. 2

A. Distal portion of 13-day normal femur cultured in the disarticulated state for2 days, x 32.B. Distal portion of 13-day luxoid femur cultured in the disarticulated state for2 days, x 32.C. Longitudinal section of 13-day luxoid femur cultured for 2 days. Note zonesof 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. Notemarrow cavity in center of diaphysis. x 30.G. 14-day luxoid tibia cultured in the disarticulated state for 2 days. Note lack ofmarrow cavity, x 30.

Luxoid skeletal defects 353observed in vivo. The distal portion of the normal tibia begins to develop amalleolus, which is lacking in the luxoid tibia. The luxoid fibula becomesthickened, 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 headand trochanters. Distally, condyles are barely distinguishable on the normalfemur, but are absent from the luxoid femur. Histologically, the 14-day normaland luxoid femora show a large central zone of hypertrophy and uniform rowsof small flattened cells which constitute the zone of proliferation. Whereasnumerous basophilic osteoblasts occur along the inner layer of the normalperiosteum, osteoblasts are relatively sparse in the luxoid periosteum. Also,a faint bone collar can be identified in the normal femora, but is lacking in theluxoid rudiments. Osteoblastic activity in the normal tibia and fibula lags behindthat in the normal femur. The luxoid counterparts of these elements likewiseshow retardation.

After 2 or more days in culture in the articulated state, typical normal andluxoid features develop, respectively, for each element. The disarticulatedanlagen maintained 2 days in culture likewise show differences from one another(Fig. 2D, E). Where prominent condyles develop on the normal femur the distalend of the luxoid femur develops a knob-like expansion which lacks an inter-condylar fossa. The normal tibia shows slight condyle formation and tapersdistally to form a malleolus. In contrast, the luxoid tibia is retarded and lacksany 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 ofa marrow cavity. In the cultured normal tibiae, cavitation can be distinguishedin the diaphyses of gross and histological specimens (Fig. 2F). The luxoidelements lack any marrow cavity at this stage, although zones of hypertrophyand proliferation are evident (Fig. 2G). Normal and luxoid elements shownumerous basophilic osteoblasts along the inner layer of the periosteum.A periosteal bone collar, however, is still absent in the luxoid rudiments. In thenormal 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 limbare well developed. The normal femur exhibits prominent condyles distally, aswell as a large and distinct marrow cavity in the diaphysis. Although the distalend of the luxoid femur is expanded, an intercondylar fossa is lacking. Marrowcavities are beginning to form in the luxoid anlagen at this stage. After 2 ormore days in culture in the articulated state the elements continue to showfeatures consistent with their genotype (Fig. 3 A, B).

When disarticulated elements are maintained in vitro for 2 days the luxoidrudiments lag behind the normal ones with respect to the size of the marrowcavity. The luxoid tibia, in particular, shows only a narrow band indicating

Luxoid skeletal defects 355slight cavitation (Fig. 3C). In the normal and luxoid specimens a bone collarcan be detected beneath the osteogenic layer of the periosteum. Distinct zonesof hypertrophy and proliferation are also easily identified.

Chorioallantoic explanationBecause of the technical difficulties involved in explantation onto the chorio-

allantoic membrane (CAM) of the chick, fewer elements were obtained thanwas the case with the grid method. A total of 45 disarticulated skeletal rudimentswas successfully maintained on the CAM. Within 24 h after explantation theseelements were penetrated by blood vessels from the host. In each instance theperiosteum was invaded by numerous small vessels, after which a larger vesselwould penetrate the diaphysis as a periosteal bud.

In all age groups gross features characteristic of normal and luxoid elementsmanifest themselves after two or more days on the CAM. In the normal femurthe distal portion becomes expanded and shows well-defined condyles, whereasthe distal portion of the luxoid femur merely develops a club-shaped expansion(Fig. 3D, E). The tibiae likewise develop according to their genotype. Thenormal tibia shows an enlarged epiphysis proximally and a tapered malleolusdistally (Fig. 3F). Despite an advantageous, well-vascularized position on theCAM, the luxoid tibia develops as a slender, rudimentary element (Fig. 3G).

Histologically, 13-day normal and luxoid femora cultured for 2 days showwell-defined zones of hypertrophy, proliferation and reserve cartilage, as well asthe beginning of a periosteal bone collar (Fig. 3H). When these elements aremaintained 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 cavitydevelops; however, the degree of calcification and cavitation in the luxoidelements is retarded.

FIG. 3

A. 15-day normal femur cultured in the articulated state for 2 days. Well-developedcondyles are present, x 30.B. 15-day luxoid femur cultured in the articulated state for 2 days. Normal condylesare 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 stateon the CAM for 2 days, x 90.


After 2 days on the CAM histodifferentiation of the 14-day rudiments isexcellent. In the normal femur the greater portion of the diaphysis consists ofgreatly hypertrophied chondrocytes, although calcification is not as yet present.Peripheral to these are wide bands of proliferation. Osteoblasts now form acontinuous layer along the periosteum, beneath which is the bone collar. Theluxoid femur exhibits a large central zone of hypertrophy, peripheral zones ofproliferation, numerous osteoblasts and a slight bone collar. As developmentproceeds beyond 2 days on the CAM normal and luxoid elements continue todifferentiate, and a marrow cavity develops in each rudiment. In the luxoidrudiments, however, the extent of ossification always lags behind that seen inthe normal ones.

DISCUSSION

The results of this study indicate that skeletal elements isolated from the hindlimb of the luxoid mouse embryo at 13 or more days' gestation have the capacityfor self-differentiation in vitro. At 13- to 131-day s of gestation chondrification ofthe luxoid tibia is considerably retarded in vivo. The luxoid femur and fibula areretarded to a lesser degree, although they lag behind their normal counterpartswith respect to the establishment of the various cartilaginous zones, as indicatedhistologically by the smaller central areas of hypertrophy seen in the luxoidelements. When these are disarticulated, virtually stripped of surroundingmesenchyme and cultured for 2 or more days, they continue to exhibit the lagin chondrogenesis which occurs in vivo. This retardation cannot thus be attri-buted to factors in the intact limb which might suppress normal growth anddifferentiation of the skeletal elements.

In luxoid limb rudiments the formation of a bone collar also lags behind thatseen in the normal condition. The bone collar can be identified in normal 14-to 14|-day femora but is lacking in their luxoid counterparts. With the gridmethod of culture used in the present study the bone collar is maintained innormal elements, but it does not show any increase in size. If the collar is notpresent at the time of explanation it fails to develop in culture.

With the CAM method, however, the collar develops in both normal andluxoid elements, including those which lack any indication of a collar at thetime of explantation. Development of a marrow cavity appears to be dependentupon the presence of vascular elements, since well-developed cavities becomeapparent regardless of whether or not cavitation has begun at the time therudiments are explanted. The luxoid elements, however, show retardation in theextent of cavitation in comparison with normal elements of the same age. Incontrast to the CAM technique, the grid method of culture does not seem to beconducive to the establishment of a marrow cavity. Chick rudiments cultured bythe 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 357trophy before the perichondrium can become osteogenic (Fell, 1932). The lag inhypertrophy illustrated by the luxoid rudiments was always followed by a lag inosteogenic activity in the perichondrium.

According to Forsthoefel (1959), ossification of luxoid elements reaches astate comparable to that of their normal counterparts at the time of birth. Thiswas not tested with the grid method of culture because of hydration artifactsand gross abnormalities which commonly occur in mouse skeletal elementsmaintained beyond 4 or 5 days in vitro (Biggers, Gwatkin & Heyner, 1961).Although ossification takes place more readily in CAM grafts, insufficientnumbers of luxoid and normal elements were obtained from prolonged periodsin culture to verify Forsthoefel's observation under these conditions.

The morphological integrity of the skeletal elements is maintained equally wellwith the two culture techniques used in the present study. Of importance is thenature of the luxoid defect which involves a failure to form normal condyles onthe 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 thedepth of the intercondylar fossa. Interaction of Strong's luxoid gene with Green'sluxoid and Carter's luxate likewise results in abnormal development of condyles(Forsthoefel, 1962). The presence of tibial hemimelia in all of these mutants hasbeen suggested as a factor which might exert some inhibiting influence on themorphogenesis 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 asan isolate, normal condyles fail to form. This defect is thus not dependent uponthe continuous association of femur with tibia or with other tissues of the hindlimb, at least in the stage of development during which the defect becomesmorphologically distinct. Attention should thus be focused on interactionswhich may occur prior to this period.

Efforts to culture disarticulated elements of early developmental stages havenot met with much success because of technical difficulties (Griineberg, 1963).We have obtained a few 12-day rudiments which appear to be self-differentiatingin the articulated state in vitro, but isolation of early skeletal 'elements' isprecluded by the continuous mesenchymal condensation which representsfemur, tibia and fibula, and possibly some skeletal muscle as well (Forsthoefel,1963).

The sequence of mesenchymal condensations may be an important factor inthe induction of tibial hemimelia, accompanied by a thickening of the fibula.Since the blastemal condensation apparently begins postaxially, an overabundantaccumulation of postaxial mesenchyme might result in an enlarged fibula witha correspondingly reduced tibia (Forsthoefel, 1963). Our results have shownthat this predominance of the fibula is maintained in vitro, as indicated by therelatively thicker diaphysis of the luxoid fibula in comparison with that of theluxoid tibia cultured for 2 or more days.


Our results also indicate that the autonomous nature of the luxoid defect in themouse is similar to that observed for various skeletal anomalies found in thechick 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 beaffected by its genotype. As indicated in Table 2, a wide range of growth wasexhibited by normal and luxoid elements maintained in culture for 2 days.Because of insufficient numbers, however, statistical analyses were not appliedto these data. Moreover, no conclusions could be drawn concerning the capacityfor over-all growth, since no measurements were taken of wet weight or dryweight.

Morphological differentiation of a skeletal element is thought to take placeduring the procartilage and early cartilage phase of development (Fell, 1956).During this time the element does not seem to be subject to extrinsic factors, asindicated by the excellent morphogenesis of the rudiments in the grid cultureenvironment and on the CAM. Bateman (1954) suggests that there is an almosttotal lack of extrinsic control on the pattern of development of individual mousebones throughout gestation. Once a bone begins to ossify, however, it seems tobecome dependent upon the presence and availability of vascular elements, sincewe found that ossification proceeded adequately only on the highly vascularchorioallantoic membrane.

SUMMARY

1. Skeletal elements isolated from the hind limbs of luxoid (lu) and normalmouse embryos of 13-15 days' gestation maintain their morphological integrityin vitro.

2. Articulated and disarticulated anlagen of femora, tibiae and fibulaemaintain typically luxoid features when cultured with the grid method or asexplants on the chorioallantoic membrane (CAM). The gross and histologicalnature of the limb defects in the luxoid embryo are not dependent upon con-tinuous association of the femur, tibia and fibula during the stages examined inthe present study.

3. Chondrification is obtained with both culture techniques; however,ossification and the establishment of a marrow cavity are obtained only withthe CAM method.

RESUME

Developpement in vitro de deficiences squeiettiques du type 'luxoide' (lu)

1. Des elements squelettiques isoles de membres posterieurs d'embryons desouris normales et 'luxoides' (lu), du 13 au 15e jour de la gestation, conserventleur integrite morphologique in vitro.

2. Des ebauches articulees et desarticulees de femurs, de tibias et de perones

Luxoid skeletal defects 359conservent des caracteres typiquement 'luxoides' quand on les cultive selon lamethode de la grille ou en explants sur la membrane chorioallantoidienne(MCA). Les caracteres generaux et histologiques des deficiences des membres del'embryon 'luxoide' ne dependent pas d'une association continue du femur, dutibia et du perone pendant les stades examines dans ces recherches.

3. On obtient la chondrification avec les deux methodes de culture; nean-moins, 1'ossification et la formation d'une cavite medullaire ne s'obtiennentqu'avec la methode MCA.

We would like to express our appreciation to Mrs Alice Dodge and Miss Laurie Paavolafor technical assistance, and to Mr Max Millsap for the photography. This work was supportedby research grants no. HD01312-06 and HD01679-02 from the National Institute of ChildHealth and Human Development, United States Public Health Service.

REFERENCES

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BATEMAN, N. (1954). Bone growth: a study of the grey-lethal and microphthalmic mutantsof the mouse. /. Anat. 88, 212-60.

BIGGERS, J. D., GWATKLN, R. B. L. & HEYNER, S. (1961). Growth of embryonic avian andmammalian tibiae on a relatively simple chemically defined medium. Expl Cell Res. 25,41-58.

CARTER, T. C. (1951). The genetics of luxate mice. /. Genet. 50, 277-99.CENTER, E. M., HUNTER, R. L. & DODGE, A. H. (1967). Effects of the luxoid gene (/«) on

liver esterase isozymes of the mouse. Genetics, 55, 349-58.ELMER, W. A. (1968). Experimental analysis of the Creeper condition in chickens. Devi Biol.

18, 76-92.FELL, H. B. (1932). The osteogenic capacity in vitro of periosteum and endosteum isolated

from the limb skeleton of fowl embryos and young chicks. /. Anat. 66, 157-80.FELL, H. B. (1956). Skeletal development in tissue culture. In The Biochemistry and Physiology

of Bone (ed. G. H. Bourne), pp. 401-41. New York: Academic Press.FELL, H. B. & CANTT, R. G. (1934). Experiments on the development in vitro of the avian

knee-joint. Proc. R. Soc. B 116, 316-51.FELL, H. B. & ROBISON, R. (1929). The growth, development and phosphatase activity of

embryonic avian femora and limb-buds cultivated in vitro. Biochem. J. 23, 767-84.FORSTHOEFEL, P. F. (1958). The skeletal effects of the luxoid gene in the mouse, including its

interactions with the luxate gene. /. Morph. 102, 247-88.FORSTHOEFEL, P. F. (1959). The embryological development of the skeletal effects of the luxoid

gene in the mouse, including its interactions with the luxate gene. /. Morph. 104, 89-152.FORSTHOEFEL, P. F. (1962). Genetics and manifold effects of Strong's luxoid gene in the mouse,

including its interactions with Green's luxoid and Carter's luxate genes. /. Morph. 110,391-420.

FORSTHOEFEL, P. F. (1963). Observations on the sequence of blastemal condensations in thelimbs of the mouse embryo. Anat. Rec. 147, 129-38.

GREEN, M. C. (1955). Luxoid—A new hereditary leg and foot abnormality in the house mouse./ . Her ed. 46, 90-100.

GRUNEBERG, H. (1943). The development of some external features in mouse embryos./. Hered. 34, 88-92.

GRUNEBERG, H. (1963). The Pathology of Development (a Study of Inherited Skeletal Disorders).New York: Wiley.

HAMBURGER, V. (1941). Transplantation of limb primordia of homozygous and heterozygouschondrodystrophic (Creeper) chick embryos. Physiol. Zool. 14, 355-64.


HINCHLIFFE, J. R. & EDE, D. A. (1968). Abnormalities in bone and cartilage development inthe ta/pid3 mutant of the fowl. /. Embryo/, exp. Morph. 19, 327-39.

MURRAY, P. D. F. & HUXLEY, J. S. (1925). Self-differentiation in the grafted limb-bud of thechick. /. Anat. 59, 379-84.

RUDNICK, D. (1945). Differentiation of prospective limb material from Creeper chick embryosin coelomic grafts. J. exp-. Zool. 100, 1-17.

{Manuscript received 30 September 1968)

Development of luxoid (lu) skeletal defects in vitro

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