the role of movement in the development of joints and...

J. Embryol. exp. Morph. Vol. 22, 3, pp. 349-71, November 1969 3 4 9Printed in Great Britain

The role of movementin the development of joints and related structures:

the head and neck in the chick embryo

By The late P. D. F. MURRAY AND DANIEL B. DRACHM AN1

From the Department of Zoology, University of New England {Australia) and theDepartment of Neurology, Tufts-New England Medical Center, Boston, U.S.A.

Skeletal muscle contractions are necessary during embryonic life for thenormal development of joints. The general features of joint development inimmobile limbs were first studied with the techniques of grafting and organculture. (Murray, 1926; Murray & Selby, 1930; Fell, 1925; Fell & Canti,1934; Hamburger & Waugh, 1940; Lelkes, 1958). However, these methods ofnecessity entail a drastic alteration in the environment of the developingarticulations, which may result in gross distortions of the skeletal structuresthemselves. More recently, neuromuscular blocking agents have been used toproduce paralysis of chick embryos in ovo. When administered intravenously,these pharmacological substances produce profound paralysis, which may becontinued for prolonged periods during embryonic development (Drachman &Coulombre, 1962a, b).

Drachman & Sokoloff (1966) have analyzed the primary development ofthe knee, ankle and toe joints of the chick embryo by the use of these methods.In these relatively simple hinge-type joints, paralysis prevents the initialformation of cavities, although morphogenesis of the joint regions proceedsup to the point when cavitation should occur. Eventually the articular surfacesbecome united by fibrous bonds or by cartilaginous 'fusion'. The form of thearticular surfaces and of the accessory structures is altered in the paralyzedlimbs. In a separate series of experiments Murray & Smiles (1965) found thatpharmacologically produced paralysis prevents the development of' adventitiouscartilage' which normally appears at the articular surfaces of certain membranebones. Both of these studies were concerned with the role of movement in theinitial formation of only a few types of articular structures. The presentinvestigation was undertaken to determine the effects of prolonged paralysison the morphogenesis of a greater variety of articulo-skeletal structures. Forthis purpose the joints, cartilages and skeleton of the head and neck were

1 Author's address: Dept. of Neurology, Johns Hopkins School of Medicine, Baltimore,Maryland, 21205, U.S.A.

350 P. D. F. MURRAY AND D. B. DRACHMAN

studied in chick embryos which had been paralyzed from the 7th through the19th days of incubation with botulinum toxin. The head and neck are particularlywell-suited for this type of study, since they include virtually the entire rangeof types of articulations and accessory articular structures.

MATERIALS AND METHODS

Immobilization was produced by the injection into a chorioallantoic veinof type A crystalline botulinum toxin (kindly supplied by Dr E. Schantz, FortDetrick, Maryland). The toxin, which was stored at 3 °C in acetate buffer, wasfreshly diluted in chick embryo Ringer's solution (Rugh, 1961) immediatelybefore use. Each 0-1 ml volume of the solution contained approximately 50 /igof toxin, an amount which represents more than 10000 lethal doses for hatchedchickens. The diluted toxin was injected directly into the chorioallantoiccirculation of the experimental embryos by means of the following technique,described elsewhere in detail (Drachman & Coulombre, 1962a): a rectangularwindow was removed from the shell and shell membrane overlying the embryo,to permit access for in jection and observation. A specially designed microcatheterwas inserted within a chorioallantoic vein, under a binocular dissecting micro-scope. Each injection of 01 ml was made with a micrometer-driven syringeattached to the catheter. Between injections, the openings in the shells weresealed with cellophane tape.

Sixteen White Leghorn embryos were used in these experiments. Eachembryo received four injections, on the 7th, 10th, 13th and 16th days ofincubation. They were incubated at 37-7 °C in a humidified forced-draft incu-bator. All embryos included in the results were alive and in good condition,although paralyzed, at the termination of the experiment. They were killedby decapitation on the 19th day of incubation, and their heads and necks werefixed in 10 % formol-saline.

The material was processed as follows: (a) eight specimens were treated with1 % KOH, stained v/ith Alizarin Red S, cleared in glycerol, and dissected;(b) the remaining specimens were embedded in paraffin wax and sectionedserially at 10 ju so as to include the following structures: the articular structuresof the jaws (four specimens) and the larynx and trachea (six specimens).

For comparison with normal structures, untreated 18- and 19-day embryoswere used, after fixation in formol-alcohol, formol-saline or Susa's fluid.

The sections were stained by several different methods including the hema-toxylin, alcian blue and chlorantine red method of Lison (1954), by whichcartilage is stained blue-green, bone and collagen fibres brilliant red; Masson'strichrome method; Orcein; and Ehrlich's hematoxylin and eosin.

Development of joints 351

RESULTS

/. General findings, gross description

The 19-day embryos injected with botulinum toxin were smaller and lighterin weight than the controls. They were approximately equal in size to untreated18-day embryos, but otherwise had matured normally for their age, and hadno bizarre malformations. Externally they showed severe fixation of multiplejoints, and protrusion of the tip of the lower beak beyond the upper. The facialpart of the skull was narrowed, while the cranial part was of normal width.All of the skeletal muscles of the body were strikingly shrunken and fatty. Theskin, feathers, subcutaneous tissues and internal organs were unremarkable,and the yolk sac was partially retracted into the abdominal cavity, as occursnormally on the 19th day (Romanoff, 1960).

//. Articular cavities

The outstanding finding in the paralyzed embryos was the nearly completeabsence of cavities in articulations of all varieties. The areas in which cavitieswould normally be found were occupied by other tissues, more or less firmlybinding the articular elements together.

(a) Articulations of the head. The skulls of birds are said to be 'kinetic', sinceboth the upper and lower jaws move relative to the cranium (Bellairs & Jenkins,1960). Such mobility derives from a complex apparatus of bones, muscles andjoints, which is characteristic of most birds, but is not present in mammals.The chief pivot of this apparatus is the quadrate bone, which articulates withthe bones forming the upper and lower beaks as well as with the cranium(Fig. 1 A). The quadrate is involved in six articulations: two with the pterygoidbone, and one each with the mandible (at Meckel's cartilage), the quadratojugal,squamosal and pro-otic bones. The pterygoid, in turn, articulates mediallywith the parasphenoid (actually, the parasphenoid-basisphenoid) and anteriorlywith the palatine. Three different types of movements are possible at thesejoints: rotation (quadrate-squamosal), hinge action (quadrate-mandibular), andgliding (all of the remaining joints) (Chamberlain, 1943). The eight pairs ofmobile joints have been studied in serial sections of the skulls of four botulinum-treated embryos.

Normally, all of these diarthrodial joints have articular cavities. In theparalyzed embryos, such cavities were nearly always absent (Fig. 3A, B, C).In their place, fibrous, cartilaginous or bony tissue joined the articular elements(see section on fusion, p. 357). In most instances, the locations of the presumptivejoints were identifiable. However, little or no trace of the articulations betweenthe quadrate and pterygoid bones could be detected. Only three of the 64 jointsstudied contained spaces resembling normal cavities; two of these occurred atpterygo-parasphenoid joints, and one at a pterygo-palatine joint. The possiblesignificance of these three exceptional spaces is considered below.


Q.J.-J.-M.

Aland.

Artie. Q.

Artie. M.Pter.

1 cm

Pa/

QJ.-J.-J

Otic Q.

ParaOrb. Q.

R.P.M.-Mand.

Pter.Quad.

LEGENDS FOR LINE DRAWINGS—FIGS. 1 AND 2

Abbreviations used: Artie. M. = Articular facet of Meckel's cartilage; Artie. Q. =Articular process of the quadrate bone; Mand. = Mandible; M.P.M. = Medial process ofMeckel's cartilage; Orb. Q. = orbital process of the quadrate bone; Otic Q. = Otic processof the quadrate bone; Pal. = Palatine bone; Para. = Parasphenoid bone; Pter. - Pterygoidbone; Q.J. = Quadratojugal bone; Q.J.-J.-M. = Quadratojugal-jugal-maxillary arcade;Quad. = Quadrate bone; R.P.M. = Retroarticular process of Meckel's cartilage; V. —Vomer bone.

Fig. 1 A. The posterior part of the skull of a normal 18-day chick embryo, seenfrom the left. B. Corresponding region of a paralyzed 19-day chick embryo. Note thefusion of Meckel's cartilage with the articular process of the quadrate. The retro-articular process of the mandible is smaller in the paralyzed embryo, and lacks theupward angulation. Drawings prepared by tracing from photographs of KOH-Alizarin-glycerol preparations.


(b) Articulations of the cervical spine. Serial sections were made of the necksof five paralyzed embryos, three in the sagittal plane and two in the transverseplane. Each specimen included three to ten vertebrae. A series of normalembryos from 14 to 19 days of incubation age were also studied.

In the normal chick, the mobile joints of the neck include those between theatlas, axis and occiput, at which gliding and/or pivoting motions occur; thosebetween the vertebral bodies (intercentral), which permit gliding and hingeaction; and those between the vertebral articular processes (interneural), which

Para.Pter.

Orb. Q.Artie. Q.

Q-J.

Otic Q.

Mand.Q.J.-J.-M.

1 cm

Fig. 2 A. Ventral view of the posterior part of the skull of a normal 18-day embryo,with the mandible removed. B. Corresponding region of a paralyzed 19-dayembryo, with the mandible still attached. Note the overall narrowness of the skull,compared with the normal. All of the bones are present in the paralyzed specimen,although their shapes and positions are altered from the normal. See text.

are capable of gliding movement (Chamberlain, 1943). Normally, all of thesejoints contain well-formed cavities. In the paralyzed embryos, cavities wereinvariably absent at the articulations of the vertebral bodies, the atlas, axisand skull. These structures were solidly fused from end to end (Fig. 4A).Small clefts or interrupted lines, which were often seen between adjacentvertebral centra, presumably represented the sites of the original interzones.

The 'interneural' articulations were ill-defined, since the pre- and especiallypost-zygapophyseal processes were poorly developed. In some specimens, thepostzygapophyses were absent. However, small articular areas were foundalong the zones of contact of successive neural arches. These articulations

23 J E E M 22


Orb.Q.


were undoubtedly functionless, because of the rigid fusions of the vertebralbodies. Some, but not all of the contiguous neural arches were fused.

(c) Larynx and Trachea. The larynx and trachea were studied in serialsections of three 18-day normal embryos, and five botulinum-treated specimens.In the normal embryos, diarthrodial joints were present at three sites in thelarynx: (1) between the ventral process of the arytenoid and the lateral com-ponent of the cricoid; (2) between the dorsal and lateral components of thecricoid and (3) between the ventral component of the arytenoid and the dorsalcomponent of the cricoid. Well-formed articular cavities were found at thefirst of these joints in all the embryos studied, while cavitation was variableat the other locations.

In the paralyzed specimens, none of the articulations had patent cavities.Fibrous or cartilaginous fusion was invariably present, sometimes obscuringthe originally separate origin of the laryngeal cartilages.

The tracheae of birds, like their necks, are very mobile, and since the rings

EXPLANATIONS OF FIGS. 3 AND 4

Abbreviations used: Art. Pr. Q = Articular process of the quadrate; /. Con. = Innercondyleof the quadrate; L. Con. = lateral condyle of the quadrate: M. = Meckel's cartilage;M.P.M. = Medial process of Meckel's; M. Int. Add. = Internal adductor muscle of themandible; Odont. = Odontoid process of the axis vertebra; Orb. Q. - Orbital process ofthe quadrate bone;Para. = Parasphenoid bone; Pter. = Pterygoid bone; Q.J. = Quadrato-jugal bone; Sa. = surangular bone; Sa. Adv. Cart. = Adventitious cartilage of surangularbone.

Fig. 3 A. Part of a coronal section through the quadrate and related bones of theskull of a normal 18-day-old chick embryo, for comparison with Figs. B and C.Note the well-developed joint spaces separating the quadrate from Meckel'scartilage and the quadratojugal. Neither the orbital process of the quadrate northe medial process of Meckel's lies in contact with the parasphenoid bone. Thesurangular bone has well-developed adventitious cartilage. The open spaces withinthe bones in this and Figs. B and C are pneumatic cavities.Fig. 3B. A coronal section from a paralyzed 19-day-old embryo, through an areacomparable to that shown in A. Note the absence of an articular cavity and thefusion of the articular process of the quadrate and Meckel's cartilage. The orbitalprocess of the quadrate is distorted in such a way as to indent the parasphenoid,with which it is fused. The pterygoid bone is closely attached to the quadrate andparasphenoid bones. The surangular bone lacks adventitious cartilage.Fig. 3C. A coronal section from a paralyzed 19-day-old embryo through thequadrate and related bones. This section shows striking distortion of the quadratebone with marked widening of its intercondylar region. The medial process ofMeckel's cartilage is brought into abnormal contact with the parasphenoid. Thissection also shows the absence of the joint cavity between the quadrate and Meckel's,and indentation of the parasphenoid by the orbital process of the quadrate, as in B.Fig. 3D. From a paralyzed 19-day-old embryo. The quadratojugal (above) articu-lating with the quadrate. Note the absence of adventitious cartilage which shouldnormally be present on the quadratojugal.Fig. 3E. From a paralyzed 19-day-old embryo. The cartilaginous head of the oticprocess articulating with the bony squamosal. Adventitious cartilage, whichshould normally be present on the squamosal is absent. There is no joint cavity.

23-2


are so close together, they inevitably move upon one another. A series ofbursae, located laterally or ventro-laterally between adjacent tracheal rings,appeared to be situated so as to facilitate free motion of the trachea (Fig. 4E).The bursae are sometimes lined by flattened mesothelial cells.

In the paralyzed embryos, the bursae were absent. The tracheal rings werejoined by fibrous connective tissue or cartilage (Fig. 4F, G).

///. Fusion

In the normal embryos, fusion never occurred at any of the mobile articulationsof the skull, cervical spine, larynx or trachea. Points of contact between articu-lating skeletal elements were separated by joint cavities and/or other specializedarticular structures. In the paralyzed embryos, fusion regularly took placeacross the presumptive joint regions, and at other sites where skeletal elementswere in contact. The fusing tissue was cartilaginous, bony or fibrous, dependingin part on the composition of the elements being fused. Many examples of eachtype of fusion were present in the material from the botulinum-treated embryos.

(a) When the contiguous elements were both cartilages, they were fused bycartilage or occasionally by fibrous connective tissue. In our material, suchcartilage-cartilage interfaces were present at articulations between any two'permanent cartilages', or any two 'replacement bones'.

The permanent cartilage rings of the trachea were most often fused dorsaUyin the mid-line, although fusion also occurred ventrally or laterally (Fig. 4F, G).In one specimen, all of the 29 rings present were fused mid-dorsally, makinga long unbroken cartilaginous rod from which the unfused parts of the rings

Fig. 4A. A sagittal section through the cervical vertebrae 3-5 of a paralyzed19-day-old embryo. The centra are fused by cartilage. Hypapophyses are absent.Fig. 4B. A coronal section from a paralyzed embryo, showing the pterygoids fusedto the parasphenoid, without an articular cavity or adventitious cartilage.Fig. 4C. A transverse section through the atlas vertebra of a normal 18-dayembryo. Note the odontoid process lying in a groove separated from the atlas.Compare with Fig. D.Fig. 4D. A transverse section through the atlas vertebra of a paralyzed 19-day-oldembryo. Note the fusion of the odontoid process with the body of the atlas.Fig. 4E. A parasagittal section through the trachea of a normal 19-day chickembryo, showing two mesothelial-lined bursae lying between three adjacent trachealrings.Fig. 4F. Part of a sagittal section through the trachea of a paralyzed 19-day embryo,showing in section the dorsal and ventral portions of the rings. The dorsal andventral walls are brought artificially close together to save space. Dorsally, therings are fused with one another, but the continuous cartilaginous rod whichresults shows periodic thickenings which must represent the positions of originallyseparate rings. Fusion is not seen ventrally.Fig. 4G. Sagittal section through the anterior part of the trachea of a paralyzedembryo. In the roof (left) of the trachea the rings are completely fused to forma continuous rod, without even the periodic thickenings seen in Fig. F. Unfusedrings are apparent ventrally, but no bursae are present.


projected on each side, like ribs from a backbone. The trachea never becameenclosed in a cartilaginous tunnel as it would have been if all the rings hadfused completely. The cartilages of the larynx were fused by cartilage, oroccasionally by connective tissue. In some cases the cartilaginous fusion wasso complete as to obscure the site of the original articulations.

Another instance of cartilaginous fusion of two 'permanent' cartilagesoccurred at the articulation between the epi- and pharyngo-branchial cartilages,which is normally a syndesmotic, or fibrous joint.

Since the articular ends of replacement bones were composed of 'primary'cartilage in paralyzed as well as normal embryos, they behaved like othercartilages with respect to fusion. In the heads of the experimental embryos,cartilaginous fusion occurred at the quadrato-mandibular and quadrato-pro-otic joints (Fig. 3B, C). The skeletal elements which participate in thesearticulations (i.e. the quadrate and pro-otic bones, and the Meckel's cartilageportion of the mandible) are replacement bones. In the cervical spine, thevertebral bodies, which are also replacement bones, were fused by cartilage(Fig. 4A). The atlas and axis vertebrae were joined by cartilaginous bridgesventrally, and the odontoid process of the axis was fused to the floor of theatlas by cartilage (Fig. 4C, D).

At the site of these fused articulations between replacement bones, cartilageextended across the presumptive joint regions, establishing continuity betweenthe opposed elements. Sections often showed traces of the original joint inter-zones in the form of lines or small clefts partially breaking the continuity ofthe cartilage. In some specimens, loose connective tissue occupied the regionsadjacent to the cartilage bridges.

(b) When the contiguous elements were both bones, they were fused by boneor by fibrous connective tissue.

In normal embryos, two bones never articulate directly with one another,since their articular surfaces are always covered by cartilage. However, in theparalyzed embryos, membrane bones failed to develop 'secondary' cartilageson their articular surfaces (see p. 359). At articulations between two membranebones, the bare bone surfaces faced each other directly. Such articulations werefused by bone or fibrous connective tissue in the botulinum-treated embryos.Thus, bony fusion was present in three of the four sectioned specimens at thepterygo-parasphenoid joint (Fig. 4B), while the fourth was fused at a pointposterior to the usual site of articulation. The pterygo-palatine joint wasfused by bone in one case, and by connective tissue in two of the three othersectioned cases.

It is of particular interest that the neural arches of the atlas and axis vertebrae,which develop as membrane bones, were fused by bone. In contrast, theventral portions of the same vertebrae, which originate as replacement bones,were fused by cartilage.

In the paralyzed embryos, distortions of certain skeletal elements resulted


in the apposition of parts of bones which do not normally make contact. Bonyor fibrous fusion took place at these extra-articular sites. Thus, the articularand orbital processes of the quadrate, and the pterygoid, were fused with theparasphenoid bone in many of the experimental embryos (Fig. 3B, C).

(c) When one of the contiguous elements was cartilage and the other wasbone, fusion took place by fibrous connective tissue. Three examples of fusionof heterogeneous skeletal elements were found in the experimental material,i.e. at the articulations between the quadrate (a replacement bone), and thequadratojugal, pterygoid and squamosal bones (which are all dermal bones)(Fig. 3B-E).

It was particularly striking that the quadrato-squamosal joint was alwaysfused by fibrous tissue, while the immediately adjacent articulation betweenthe quadrate and the pro-otic (a replacement bone) was fused by cartilage.

There are normally two small articulations between the quadrate and thepterygoid, and a narrow duct which passes between the pneumatic spaces inthe two bones at a site distinct from either joint. In the paralyzed embryosthe only remnant of the two articulations was a fibrous band connecting theend of the pterygoid to the quadrate. However, fibrous connective tissue andoccasionally bony trabeculae surrounded the pneumatic duct, and helped toanchor the two bones together. This periductal fusion falls into the groupdescribed above for extra-articular sites.

IV. Secondary cartilage formation

In the normal chick, 'adventitious' cartilages were present at the articularsurfaces of membrane bones, in the eight locations previously described byMurray (1963). In the paralyzed embryos, adventitious cartilage was invariablyabsent (Fig. 3A-E). Furthermore, the late-forming 'articular' cartilage padsnormally found at the pterygo-parasphenoid joint were missing.

In contrast, the primary cartilage of replacement bones, trachea and larynxwas present and histologically normal in the experimental embryos, indicatingthat the botulinum toxin treatment did not directly inhibit chondrogenesis.

V. Distortions of the skeleton

Although the general appearance of the head and neck was remarkablynatural in the experimental embryos, the skeletal elements were distorted inimportant details. This section contains a descriptive account of examples ofsuch distortions which occurred consistently in the experimental material.

(a) Mandible. In all of the paralyzed embryos the tip of the mandibleprotruded beyond the upper beak, by an average of 1-9 mm (Fig. 5). In orderto determine whether the upper beak was abnormally short or the lower wasabnormally long, several measurements in paralyzed and 19-day-old normalembryos were made: (1) from the tip of each beak to the 'gape' or openingof the jaws; (2) from the tip of each beak to the ext. auditory meatus; (3) from


the tip of the upper beak to the posterior border of the skull; (4) from the tipof the lower beak to the base of the retroarticular process of the mandible(i.e. mandibular length).

The results are presented in Table 1. Regardless of the parameter measured,the upper beaks were shorter and the lower beaks longer in the paralyzedthan in the normal embryos. Possible explanations of these findings arepresented in the discussion.

Imm

Fig. 5. The head of a paralyzed embryo, to show the underhang of the mandible.

Table 1. Average measurements of beak length

UnderhangUpper beak lengthMandibular lengthRetroarticular process of mandibleTip to 'Gape' upper

lowerTip to EAM upper

lower

* 5 specimens.

DistancesA/

19-daynormal*

025-6419-652-821-21192-332-30

t 8 specimens

in mm

Botulinum-treatedj

1-924-5420-31

2-571091-242-22-38

T\'(f

L/inerence(Botulinumminus normal)

1-9- 0 1

0-66-0-25- 0 1 1

005- 0 1 3

008

(b) MeckeVs cartilage. The retroarticular process of Meckel's cartilageextended straight backwards in the experimental embryos, rather than beingangled upwards, as in the normal. It was both shorter and slighter than in thenormal (Fig. 1 A, B).

In normal embryos, the medial process of Meckel's cartilage did not comeinto contact with the floor of the cranium. In the paralyzed specimens, the tipof the medial process of Meckel's was sunken into deep or shallow indentationsin the parasphenoid bone in every specimen (Figs. 2B; 3C).


(c) Quadrate. In normal embryos, the quadrate bears two accessory structuresknown as the 'condyle' and the 'stop' at its articulation with the quadratojugal(Murray, 1963). The posterior end of the quadratojugal passes over the 'condyle',curving ventrally behind it, between it and the 'stop'. The 'stop' is thoughtto prevent backwards dislocation at the joint. In the paralyzed embryos bothof these accessory structures were absent, although the posterior end of thequadratojugal occupied approximately its normal position.

The orbital process of the quadrate made abnormal contact with the para-sphenoid bone at the base of the cranium, which it indented and fused within many cases (Fig. 3B, C).

(d) Cervical spine. In the paralyzed embryos, the cervical spine was bent

1 cm0-5 cm

10

Rostral Caudal

Fig. 6. Drawings prepared by tracing from photographs. A. The first eleven vertebraeof the neck of a normal 18-day embryo, in dorsal view. KOH-Alizarin-glycerol.The apparent gap in the atlas indicates a mid-dorsal region of its neural arch inwhich ossification has not yet replaced the cartilage. The vertebral column is neitherbent nor twisted. Stippled areas represent the vertebral centra seen from dorsallythrough the gaps between the neural arches. Neural arches three and four arepierced by foramina for the dorsal divisions of spinal nerves. Notice the narrowneural arches; compare Fig. 6B. Numerals indicate the serial numbers of vertebrae.B. The first ten vertebrae of the neck of a paralyzed embryo, in approximatelydorsal view. Because of the torsion of the specimen, its rostral and caudal partswere photographed separately, after arrangement of each part to bring its dorsalsurface into view. In the rostral portion (left), vertebrae one to three are seen some-what from the left. In the caudal portion (right) vertebra five is seen from dorsally,vertebrae six to nine slightly from the right, and vertebra ten from dorsally. Theline of demarcation between the fused atlas and axis is indicated by a dotted line, inthe position of an apparent seam where the two neural arches join. The mid-dorsalgap in the neural arch of the atlas, and similar gaps in the axis, indicate areas ofincomplete ossification. In vertebrae three, four, five and ten similar mid-dorsallines of incomplete ossification are indicated. No attempt is made to show the ventralparts of the vertebrae because of the limited size of the mid-dorsal intervertebralapertures, and because the fusion of all centra makes the floor of the spinal canalfeatureless.


laterally, and rotated about its long axis. The bend was either to the right orto the left, and the rolation was either clockwise or counter-clockwise. (Fig. 6B).

In the normal embryos hypapophyses were present on vertebrae three toeight. These were of two sorts: on vertebrae three to five, they were mid-ventralcartilaginous protrusions from about the middle of the length of each vertebra,piercing through the perichondral bone. In vertebrae six to eight they arosefrom the epiphyseal cartilages of the vertebrae close to their anterior ends.The hypapophyses served as attachments for the cervical musculature. In theparalyzed embryos, hypapophyses of both varieties were entirely absent(Fig. 4 A).

VI. The muscles and fibrous structures especially related to muscles

The limb muscles of chick embryos paralyzed with botulinum toxin andother neuromuscular blocking agents have been described in detail elsewhere(Drachman, 1968).

The following muscles were studied in the present investigation:

Depressor of the mandible (m. depressor mandibulae) connecting the squamosalto the retro-articular process of Meckel's cartilage and mandibular bones.

External adductor of mandible (m. adductor mandibulae externus) from thesquamosal to the mandible.

Internal adductor of the mandible (m. adductor mandibulae internus) fromthe palatine to the medial process of Meckel's cartilage and its shaft.

External pterygoid (m. pterygoideus externus) from the pterygoid to themandible.

Quadrate-mandibular, or middle adductor of the mandible (m. adductormandibulae medius) from the orbital process of the quadrate to Meckel'scartilage and the surangular bone.

Grossly, the muscles were extremely atrophic and loose in texture, with fattyinfiltration. However, all of the muscles but one were identifiable, and couldbe traced from their normal origins to their normal insertions. The orbito-quadrate (m. spheno-pterygo-quadratus) which normally extends from theorbital region near the mid-line to the quadrate and pterygoid, could not be foundin the paralyzed embryos.

Tendons, and ligaments closely associated with muscles, were more tenuousthan in normal embryos, or were absent.

Historically, the most striking finding was the devastating reduction inmuscle bulk, with the greatest part of the muscle replaced by adipose tissue.The remaining muscle fibres were atrophic, degenerating or myotubal. In thesefibres, there was an Apparent) increase in the number of sarcolemmal nuclei,with alignment, clumping and pyknosis of nuclei. Some of the muscle fibresretained their striations while undergoing atrophy; others showed markeddegeneration, characterized by eosinophilia, swelling, and floccular changes,


with phagocytosis by histiocytes. In transverse section, some normal-sizedfibres had the characteristic appearance of myotubes with either a space ora nucleus in the centre of a ring of myofibrils. The effects on skeletal muscle ofprolonged treatment with botulinum toxin have been discussed in detailelsewhere (Drachman, 1968) and are believed to be the result of 'pharmaco-logical denervation' caused by impairment of neuromuscular transmission.

DISCUSSION

Contractions of skeletal muscle begin early in development, and assumeconsiderable prominence during embryonic life, in the many species wherethey have been studied (Angulo y Gonzales, 1935; Coghill, 1934; Kuo, 1938;Minkowski, 1920; Tuge, 1931; Windle & Griffin, 1931). In the chick, somaticmovements begin at 3̂ - days of incubation, and increase thereafter in frequencyand duration, until, at 13 days they occupy 80 % of the embryo's time (Ham-burger & Balaban, 1963; Hamburger, Balaban, Oppenheim & Wenger, 1965).That these movements play an important role in the development of articularand skeletal structures has been demonstrated by previous studies (Murray,1936; Murray & Selby, 1930; Fell & Canti, 1934; Hamburger & Waugh, 1940;Lelkes, 1958; Drachman & Coulombre, 19626; Drachman & Sokoloff, 1966;Sullivan, 1966). Recently we have used a variety of neuromuscular paralyzingagents in order to define the precise role of movement in the development ofsimple types of joints and 'adventitious' cartilages (Murray & Smiles, 1965;Drachman & Sokoloff, 1966). These studies indicated that skeletal musclecontractions are essential during embryonic development for the initial formationof joint cavities, development of 'adventitious' cartilages and certain sesamoidcartilages, and prevention of ankylosis of joints. The present study was under-taken to investigate the effect of more prolonged paralysis on a wider varietyof articular and skeletal structures. For this purpose, immobilization of chickembryos was maintained from the 7th through the 19th days of incubation bymeans of botulinum toxin, which proved to be the most effective and con-venient paralysing agent.

Type A botulinum toxin is a pure cry stall izable protein, produced by a strainof the anaerobic bacterium, Clostridium botulinum. It causes long-lastingparalysis of skeletal muscle by preventing the release of the neurotransmitter,acetylcholine. It leads to death in most animals by respiratory paralysis(Lamanna, 1959). However, since the chick embryo respires by passive gasexchange across the chorioallantoic membrane, up to the 20th day of incubation(Romanoff, 1960) it readily survives large doses of botulinum toxin or otherneuromuscular blocking agents.

Apart from muscular atrophy, which has been discussed in detail elsewhere(Drachman, 1968), the abnormalities in the botulinum-treated embryos wereconfined to the skeletal system and related integument. The effects of botulinum


toxin on skeletal and articular structures, like those of other neuromuscularblocking agents, have been shown to be due to paralysis per se, rather than tosome unrelated toxic; action (Drachman & Sokoloff, 1966). The outstandingchanges in the present material included: (1) absence of joint cavities; (2)fusion of joints and some non-jointed structures; (3) absence of adventitiousand 'articular' cartilages; (4) skeletal distortions.

These abnormalities are most simply explained on the basis of lack ofmechanical activity of muscle.

(1) The absence of joint cavities

In paralyzed embryos, nearly all of the articular cavities which are normallypresent in the head and neck were absent. This general rule held true regardlessof what the normal mechanical action of the joint might be. With rare exceptions,all of the sliding, pivoting, rotating and hinge-type joints of the head and necklacked cavities. Even the diarthrodial articulations between the laryngealcartilages and the bursal sacs between the tracheal rings were without jointspaces. In only three of the many joints studied were well-formed cavitiespresent. Incomplete joint clefts were also seen along the lines of fusion ofadjacent vertebral centra.

These observations confirm and extend previous published reports in whichimmobilization of the relatively simple joints of the lower limbs of chickembryos resulted in failure of cavity formation (Murray, 1926; Murray &Selby, 1930; Fell & Canti, 1934; Hamburger & Waugh, 1940; Drachman &Sokoloff, 1966). It is now clear that cavitation is impaired in all diarthrodialjoints irrespective of their normal mechanical action, or of the origin andcomposition of the articulating elements.

The question of why a few well-formed, and a larger number of fragmentary,joint spaces do appear in spite of muscular paralysis, is not settled. Threepossible explanations have been offered (Drachman & Sokoloff, 1966): (a)skeletal growth may produce mechanical stresses which pry open joint spaces;(b) contractions of the smooth muscle of the amnion may impart motion to thejoints, leading to cavitation; (c) maturing synovial cells may possibly differentiatein the absence of movement, and form small or partial clefts. The normal roleof movement appears to be one of rupturing the thinned mesenchymal tissuebetween these tiny spaces, thus permitting them to become confluent.

(2) Fusion

The opposing articular surfaces of paralyzed joints were bound togetherby fibrous tissue, cartilage or bone. Which of these tissues fused a given jointdepended in part on whether the articulating surfaces were composed of cartilageor bone. Any two opposing surfaces might be bound by fibrous connectivetissue. However, cartilaginous fusion occurred only between two cartilagesand bony fusion only between two bones (Fig. 7). Thus, for example, permanently


cartilaginous structures such as the larynx and trachea were more or lessfirmly fused by either cartilage or fibrous tissue. Similarly, articulations betweentwo temporarily cartilaginous (replacement) bones were found to be fusedby either cartilage or fibrous tissue. Examples of this type of fusion werefound at the articulations between replacement bones such as the quadrateand Meckel's cartilage, the quadrate and the pro-otic, and in the neck, betweenthe vertebral centra.

Cartilage F Bone

C or F B or F

Cartilage Bone

Fig. 7. Diagram to illustrate type of tissue which fuses various combinationsof cartilage and bone. B = Bone; C = Cartilage; F = Fibrous Connective tissue.

Articulations between two bones not covered by articular cartilage werejoined either by bony trabeculae, or more often by fibrous tissue. Since articularcartilages were missing from membrane bones in the paralyzed embryos (seep. 359), joints between membrane bones such as the pterygoid and parasphenoidwere fused in this fashion. Joints between one bony and one cartilaginoussurface were almost invariably bound by fibrous connective tissue. Finally,fibrous or bony fusion occasionally developed in the paralyzed specimensbetween adjacent bones which do not normally articulate, such as the quadrateand parasphenoid.

From these findings it appears that fibrous connective tissue can form betweenimmobilized articular surfaces regardless of their cellular composition. Bycontrast, the formation of cartilage or bone in the joint interspace requirestwo opposing surfaces of the same substance, suggesting that these morespecialized tissues must be derived in some way from the articular surfaces.In a previous study, it was found that fibrous ankylosis takes place earlier thancartilaginous fusion in paralyzed limb joints (Drachman & Sokoloff, 1966). It islikely that the sequence of events is as follows:

may

Immobilization -> non-fission of cavity -> fibrous fusion -»>cartilaginous or bony union, depending on the composition of the articularelements.

The question of whether fusion is 'primary' or 'secondary' actually becomesa semantic one. 'Primary fusion' is usually taken to mean failure of a jointto separate in the first place (non-fission), while 'secondary fusion' refers tofusion of skeletal elements which were previously separate. If cavity formationis used as the criterion of separation of elements, then the immobile joints,which do not develop cavities, may be said to undergo 'primary fusion'.


Nevertheless, the cartilaginous or bony union supervenes between two elementswhich were once divided by a different type of tissue, mesenchymal or fibrous,and in this sense the; fusion may be thought of as secondary.

(3) The absence of adventitious cartilages

'Adventitious' cartilage is normally present at the articulating surfaces ofdermal bones. It appears rather late in embryonic development, about the1 lth day in the chicle (Hall, 1967), takes origin in the cambial layer of the bone,and its cells quickly become hypertrophic. In previous studies Murray (1963),Murray & Smiles (1965) and Hall (1967) have found that mechanical movementis necessary for the development and continued presence of adventitiouscartilage. Embryonic joints which were immobilized by grafting or pharmaco-logical paralysis before adventitious cartilage had appeared did not developit throughout the limited age range studied. When immobilization was begunafter initial formation of adventitious cartilage, it was gradually replaced andcovered over by bone. Finally, artificial mechanical stimulation led to develop-ment of adventitious cartilage on the quadratojugal.

In the present study, long-lasting paralysis which was begun early andmaintained throughout embryonic life resulted in permanent absence ofadventitious cartilage. This amply confirmed the principle that cartilagedevelopment in this situation is dependent on mechanical forces normallyprovided by skeletal muscle activity.

Another similar joint structure, 'articular cartilage', was also absent in theexperimental chick embryos. Articular cartilage is normally found on bothsides of the joint between the pterygoid and parasphenoid bones, and has beendistinguished from adventitious cartilage by its location and histology. It islocated in the fibrous layers of the periosteum, separated from the bone bysoft tissue, and is comprised of typical hyaline cartilage, rather than the hyper-trophic form which characterizes adventitious cartilages. The fact that 'articularcartilage' behaved like adventitious cartilage in the paralyzed embryos suggeststhat its separate classification on the basis of histological criteria may beartificial; it may be more meaningful to group it with other 'adventitiouscartilages' on the basis of their evocation by mechanical factors.

Cartilage in other situations developed normally in the experimental embryos,proving that botulhum toxin does not interfere directly with the metabolismof chondrogenic cells. It is clear from this and previous work that immobilizationproduced by a variety of methods does not prevent all cartilage formation; theprimary cartilage models of the chondrocranium, vertebrae and limbs, and thepermanent cartilages of the trachea and larynx develop independently ofmechanical action.

(4) Distortions of the skeleton

In broad outline, the skeleton of the head and neck was remarkably naturalin the experimental embryos. However, in many important details, the movable

Development of joints 2>61part of the skeleton was found to be distorted. These distortions can be groupedunder^several headings:

(a) Accessory joint structures. Murray (1963) has described a number ofknobs and protrusions adjacent to certain of the cranial articulations of thechick, which normally serve to guide and limit the motion of the articulatingbones. In the paralyzed specimens, such accessory joint structures as the'stop' and 'condyle' were absent. While the precise mode of their formationhas'not been ascertained, it appears that they depend for their development onmuscular activity.

(b) Prominences to which muscles attach. Several skeletal prominences whichnormally give attachment to muscles were either missing or grossly distortedin the experimental embryos. For example, the hypapophyses of the vertebrae,processes to which the cervical muscles attach, were entirely absent in theparalyzed specimens. Similarly the retroarticular process of Meckel's cartilage,which normally curves upwards, in line with the pull of the mandibular depressormuscle, extended straight backwards in the paralyzed embryos. Distortionssuch as these are undoubtedly due to the lack of the molding influence whichrepeated muscular pull normally exerts on skeletal structures.

(c) Malposition of skeletal elements. In many respects the anatomical relation-ships between skeletal elements were distorted in the paralyzed embryos. Someof these distortions were attributable to deformities of the bones themselves,due to one or more of the factors outlined above. Additional mechanismswhich account for certain skeletal malpositions include:

(1) Fixed retention of embryonic postures. Normally, the neck of the chickembryo is anteflexed and rotated to the right, often tucked under the right wing.When the chick is removed from the shell, the neck unkinks, because of theflexibility of the intervertebral joints. However, in the paralyzed embryos, theembryonic posture is retained, due to fixation of the joints. The vertebralcolumn of the paralyzed embryos may acquire unnatural twists, due to thepressures imposed by growth within a confined space, unopposed by muscularactivity (Sullivan, 1966).

(2) Lack of padding, due to muscle atrophy. In certain regions the skeletalmuscles' bulk is normally interposed between cranial bones, and serves asa buffer to keep them apart. For example, the space between the quadratebone and the medial wall of the orbit is normally occupied by three muscles,of which the orbito-quadratus is the largest. In the paralyzed embryos, thesemuscles were very atrophic, permitting the quadrate bone to come into contactwith the orbital wall. The lack of muscle bulk in this situation accounted for thenarrowness of the anterior skull in the experimental embryos.

(d) Protrusion of the mandible. In the paralyzed embryos, the lower beakprotruded nearly 2 mm beyond the upper, whereas in normal chicks the beaksmeet at their tips. Theoretically the appearance of a protruding mandiblemight be caused by: (a) an abnormally long mandible; (b) a short upper beak


or (c) malposition of the skeletal elements favoring a forward position of themandible. Measurements made in this study (see results) suggest that all threefactors contributed to the protrusion of the mandible in the paralyzed embryos.However, the mechanism by which a single influence, i.e. lack of muscularcontraction, produced both elongation of the mandible and shortening of theupper beak, is not at all clear. It is of interest that mice and humans withdeficiency of skeletal musculature during embryonic development showedmarked retrusion of the mandible, the reverse of the situation in chicks. Perhapsthis is a consequence of the fact that only the lower jaw is mobile in mammals,while both the upper and lower jaws are kinetic in birds.

SUMMARY

In order to study the role of muscular contractions in the development ofarticular and skeletal structures, chick embryos were kept paralyzed from the7th through the 19th days of incubation by repeated injections of botulinumtoxin. The joints, cartilages, bones and soft tissues of the head, neck, larynxand trachea were studied by dissection and histological studies in 19-day-oldparalyzed and normal embryos.

The outstanding findings included:1. Absence of joint cavities at the movable articulations of the jaws, vertebrae,

and larynx, and absence of the bursae between tracheal rings.2. Fusion across the joint regions by fibrous connective tissue, cartilage or

bone, depending on the composition of the articular elements. Fusion alsooccurred between tracheal rings, at syndesmotic joints and at other sites whereskeletal elements were in contact.

3. Absence of adventitious cartilage, normally found on membrane bonesat articulations.

4. Distortions of cartilaginous and bony structures. These distortions wereattributable to (a) failure of skeletal muscles to exert their normal moldinginfluences on these structures and (b) fixed retention of certain malpositionsresulting from growth of the embryo within a confined space, unopposed bymuscular activity.

5. Marked atrophy of the skeletal musculature and associated connectivetissue structures such as tendons and ligaments.

It is concluded that the normal development and maintenance of mobilejoints and adventitious cartilages, and the development of some features ofgross skeletal form are dependent on an active musculature.


RESUME

Le role du mouvement dans le developpement des articulations et desstructures annexes: la tete et le cou de Vembryon de poulet

Pour etudier le role des contractions musculaires dans le developpement desstructures articulaires et squelettiques, on a maintenu des embryons de pouletparalyses du 7e au 19e jour d'incubation par des injections repetees de toxinebotulique. Les articulations, les cartilages, les os et les tissues mous de la tete,du cou, du larynx et de la trachee ont ete etudies par dissection et sur coupesmicroscopiques, chez des embryons de 19 jours paralyses et normaux.

Les resultants marquants comprennent:1. Absence de cavites articulaires aux articulations mobiles des machoires,

des vertebres et du larynx et absence des bourses entre les anneaux tracheens.2. Fusion a travers les regions articulaires par du tissue conjonctif fibreux,

du cartilage ou de l'os, selon la composition des elements articulaires. Desfusions se sont egalement produites entre anneaux tracheens, aux articulationssyndesmotiques et en d'autres points ou des elements squelettiques etaient encontact.

3. Absence de cartilage adventice, present normalement sur les os de mem-brane aux articulations.

4. Distorsions des structures cartilagineuses et osseuses. Ces distorsionsetaient attribuables au fait que (a) les muscles squelettiques n'exercaient pasleur influence modelante normale sur ces structures, et que (b) certaines positionsanormales etaient fixees et conservees, provenant de la croissance de l'embryondans un espace reduit, et de l'absence d'activite musculaire pour s'y opposer.

5. Atrophie marquee de la musculature squelettique et des structures con-jonctives associees, telles que tendons et ligaments.

On en conclut que le developpement normal et le maintien d'articulationsmobiles et des cartilages adventices, et le developpement de quelques caracteresde la forme generate du squelette, dependent d'une musculature active.

This work was supported in part by grants from the Institute of Child Health andHuman Development, the N.S.W. State Cancer Council and the Australian Research GrantsCommittee.

It is a pleasure to thank Dr B. K. Hall who, besides making the measurements quoted inthe text, also read the manuscript and made many helpful suggestions, Miss Helen Macindoewho cut the sections and did much of the photographic work, Mr W. Webster of the Photo-graphic Department, and Mr Michael Webb of the Department of Zoology of the Universityof New England, and Mr Alfonse Giglio of the Tufts-New England Medical Center forfurther photographic assistance. Mr Robert Ullrich provided expert assistance with thedrawings.

Note. This study was interrupted by the sudden and tragic death of Dr Murray in the Springof 1967. The help which was generously given by Mrs. Jascha Murray and by Dr B. K. Hallhas made possible the completion of the manuscript. It is my hope that this effort maysymbolize the respect and affection of all those who knew Dr Murray. D.B.D.

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REFERENCESANGULO y GONZALES, A.. W. (1935). Further studies upon development of somatic activity

in albino rat fetuses. Proc. Soc. exp. Biol. Med. 32, 621-2.BELLAIRS, A. D'A. & JENKINS, C. R. (1960). The skeleton of birds, ch. vu. In Biology and

Comparative Physiology of Birds (ed. A. J. Marshall), pp. 241-300. New York: AcademicPress.

CHAMBERLAIN, F. W. (1943). Atlas of Avian Anatomy. Lansing, Mich.: Hallenbeck.COGHILL, G. E. (1934). Somatic myogenic action in embryos of Fundulus heteroclitus. Proc.

Soc. exp. Biol. Med. .31, 62-4.DRACHMAN, D. B. (196!?). The role of acetylcholine as a trophic neuromuscular transmitter.

In Ciba Foundation Symposium on Development of the Nervous System. London:Churchill.

DRACHMAN, D. B. & COULOMBRE, A. J. (\962a). A method for infusion of fluids into thevascular compartment of the chick embryo. Science, N. Y. 138, 144-5.

DRACHMAN, D. B. & COULOMBRE, A. J. (19626). Experimental clubfoot and arthrogryposismultiplex congenita. Lancet ii, 523-6.

DRACHMAN, D. B. & SOKOLOFF, L. (1966). The role of movement in embryonic jointdevelopment. Devi B':ol. 14, 401-20.

FELL, H. B. (1925). The histogenesis of cartilage and bone in the long bones of the embryonicfowl. /. Morph. 40, 417-59.

FELL, H. B. & CANTI, R. B. (1934). Experiments on the development in vitro of the avianknee joint. Proc. R. Soc. B, 116, 316-51.

HALL, B. K. (1967). The formation of adventitious cartilages by membrane bones under theinfluence of mechanical stimulation applied in vitro. Life Sciences 6, 663-7.

HAMBURGER, V. & BALABAN, M. (1963). Observations and experiments on spontaneousrhythmical behavior :.n the chick embryo. Devi Biol. 7, 533-45.

HAMBURGER, V., BALAEJAN, M., OPPENHEIM, R. & WENGER, E. (1965). Periodic motility ofnormal and spinal click embryos between 8 and 17 days of incubation. /. exp. Zool.159, 1-14.

HAMBURGER, V. & WALGH, M. (1940). The primary development of the skeleton in nervelessand poorly innervated limb transplants of chick embryos. Physiol. Zool. 13, 367-80.

Kuo, Z. Y. (1938). Ontogeny of embryonic behavior in Aves. XII. Stages in the developmentof physiological activities in the chick embryo. Am. J. Psychol. 51, 361-79.

LAMANNA, C. (1959). Tie most poisonous poison. Science, N. Y. 130, 763-72.LELKES, G. (1958). Experiments in vitro on the role of movement in the development of joints.

/. Embryol. exp. Morph. 6, 183-6.LISON, L. (1954). Alcian blue 8G with chlorantine fast red 5B: A technic for selective staining

of mucopolysaccharides. Stain Technol. 29, 131-8.MINKOWSKI, M. (1920). Reflexes et mouvements de la tete, du tronc, et des extremites, du

foetus humain, pendant la premiere moitie de la grossesse. C. r. Seanc. Soc. Biol. 83,1202-4.

MURRAY, P. D. F. (1926). An experimental study of the development of the limbs of thechick. Proc. Linn. Soc. Lond. 51, 187-263.

MURRAY, P. D. F. (1936). Bones: A study of the Development and Structure of the VertebrateSkeleton. London and New York: Cambridge University Press.

MURRAY, P. D. F. (196.1). Adventitious cartilage in the chick and the development of certainbones and articulations in the chick skull. Aust. J. Zool. 11, 368-430.

MURRAY, P. D. F. & SELBY, D. (1930). Intrinsic and extrinsic factors in the primary develop-ment of the skeleton. Wilhelm Roux Arch. EntwMech. Org. 122, 629-62.

MURRAY, P. D. F. & SMILES, M. (1965). Factors in the evocation of adventitious (secondary)cartilage in the chick embryo. Aust. J. Zool. 13, 351-81.

ROMANOFF, A. C. (1960). The Avian Embryo. New York: Macmillan.RUGH, R. (1961). Laboratory Manual of Vertebrate Embryology. 5th ed. Minneapolis,

Minnesota: Burgess.


SULLIVAN, G. E. (1966). Prolonged paralysis of the chick embryo with special reference toeffects on the vertebral column. Aust. J. Zool. 14, 1-17.

TUGE, H. (1931). Early behavior of embryos of the turtle, Terrapene Carolina. Proc. Soc.exp. Biol. Med. 29, 52-3.

WJNDLE, W. F. & GRIFFIN, A. M. (1931). Observations on embryonic and fetal movementsof the cat. /. comp. Neurol. 52, 149-88.

(Manuscript received 7 January 1969)

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