J. Embryol. exp. Morph. Vol. 22, 3, pp. 349-71, November 1969
349
Printed in Great Britain
The role of movement
in 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 the
Department of Neurology, Tufts-New England Medical Center, Boston, U.S.A.
Skeletal muscle contractions are necessary during embryonic life for the
normal development of joints. The general features of joint development in
immobile limbs were first studied with the techniques of grafting and organ
culture. (Murray, 1926; Murray & Selby, 1930; Fell, 1925; Fell & Canti,
1934; Hamburger & Waugh, 1940; Lelkes, 1958). However, these methods of
necessity entail a drastic alteration in the environment of the developing
articulations, which may result in gross distortions of the skeletal structures
themselves. More recently, neuromuscular blocking agents have been used to
produce paralysis of chick embryos in ovo. When administered intravenously,
these pharmacological substances produce profound paralysis, which may be
continued for prolonged periods during embryonic development (Drachman &
Coulombre, 1962a, b).
Drachman & Sokoloff (1966) have analyzed the primary development of
the 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 initial
formation of cavities, although morphogenesis of the joint regions proceeds
up to the point when cavitation should occur. Eventually the articular surfaces
become united by fibrous bonds or by cartilaginous 'fusion'. The form of the
articular surfaces and of the accessory structures is altered in the paralyzed
limbs. In a separate series of experiments Murray & Smiles (1965) found that
pharmacologically produced paralysis prevents the development of' adventitious
cartilage' which normally appears at the articular surfaces of certain membrane
bones. Both of these studies were concerned with the role of movement in the
initial formation of only a few types of articular structures. The present
investigation was undertaken to determine the effects of prolonged paralysis
on the morphogenesis of a greater variety of articulo-skeletal structures. For
this 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 the
19th days of incubation with botulinum toxin. The head and neck are particularly
well-suited for this type of study, since they include virtually the entire range
of types of articulations and accessory articular structures.
MATERIALS AND METHODS
Immobilization was produced by the injection into a chorioallantoic vein
of type A crystalline botulinum toxin (kindly supplied by Dr E. Schantz, Fort
Detrick, Maryland). The toxin, which was stored at 3 °C in acetate buffer, was
freshly diluted in chick embryo Ringer's solution (Rugh, 1961) immediately
before use. Each 0-1 ml volume of the solution contained approximately 50 /ig
of toxin, an amount which represents more than 10000 lethal doses for hatched
chickens. The diluted toxin was injected directly into the chorioallantoic
circulation of the experimental embryos by means of the following technique,
described elsewhere in detail (Drachman & Coulombre, 1962a): a rectangular
window was removed from the shell and shell membrane overlying the embryo,
to permit access for in jection and observation. A specially designed microcatheter
was inserted within a chorioallantoic vein, under a binocular dissecting microscope. Each injection of 0 1 ml was made with a micrometer-driven syringe
attached to the catheter. Between injections, the openings in the shells were
sealed with cellophane tape.
Sixteen White Leghorn embryos were used in these experiments. Each
embryo received four injections, on the 7th, 10th, 13th and 16th days of
incubation. They were incubated at 37-7 °C in a humidified forced-draft incubator. All embryos included in the results were alive and in good condition,
although paralyzed, at the termination of the experiment. They were killed
by decapitation on the 19th day of incubation, and their heads and necks were
fixed in 10 % formol-saline.
The material was processed as follows: (a) eight specimens were treated with
1 % KOH, stained v/ith Alizarin Red S, cleared in glycerol, and dissected;
(b) the remaining specimens were embedded in paraffin wax and sectioned
serially at 10 ju so as to include the following structures: the articular structures
of the jaws (four specimens) and the larynx and trachea (six specimens).
For comparison with normal structures, untreated 18- and 19-day embryos
were used, after fixation in formol-alcohol, formol-saline or Susa's fluid.
The sections were stained by several different methods including the hematoxylin, alcian blue and chlorantine red method of Lison (1954), by which
cartilage is stained blue-green, bone and collagen fibres brilliant red; Masson's
trichrome 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 lighter
in weight than the controls. They were approximately equal in size to untreated
18-day embryos, but otherwise had matured normally for their age, and had
no bizarre malformations. Externally they showed severe fixation of multiple
joints, and protrusion of the tip of the lower beak beyond the upper. The facial
part 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. The
skin, feathers, subcutaneous tissues and internal organs were unremarkable,
and the yolk sac was partially retracted into the abdominal cavity, as occurs
normally on the 19th day (Romanoff, 1960).
//. Articular cavities
The outstanding finding in the paralyzed embryos was the nearly complete
absence of cavities in articulations of all varieties. The areas in which cavities
would normally be found were occupied by other tissues, more or less firmly
binding the articular elements together.
(a) Articulations of the head. The skulls of birds are said to be 'kinetic', since
both the upper and lower jaws move relative to the cranium (Bellairs & Jenkins,
1960). Such mobility derives from a complex apparatus of bones, muscles and
joints, which is characteristic of most birds, but is not present in mammals.
The chief pivot of this apparatus is the quadrate bone, which articulates with
the 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 pterygoid
bone, and one each with the mandible (at Meckel's cartilage), the quadratojugal,
squamosal and pro-otic bones. The pterygoid, in turn, articulates medially
with the parasphenoid (actually, the parasphenoid-basisphenoid) and anteriorly
with the palatine. Three different types of movements are possible at these
joints: rotation (quadrate-squamosal), hinge action (quadrate-mandibular), and
gliding (all of the remaining joints) (Chamberlain, 1943). The eight pairs of
mobile joints have been studied in serial sections of the skulls of four botulinumtreated embryos.
Normally, all of these diarthrodial joints have articular cavities. In the
paralyzed 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 presumptive
joints were identifiable. However, little or no trace of the articulations between
the quadrate and pterygoid bones could be detected. Only three of the 64 joints
studied contained spaces resembling normal cavities; two of these occurred at
pterygo-parasphenoid joints, and one at a pterygo-palatine joint. The possible
significance of these three exceptional spaces is considered below.
352
P. D. F. MURRAY AND D. B. DRACHMAN
Artie. Q.
Q.J.-J.-M.
Aland.
Artie. M.
Pter.
1 cm
Pa/
Otic Q.
QJ.-J.-J
Orb. Q.
R.P.M.-
Para
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 of
Meckel's cartilage; Orb. Q. = orbital process of the quadrate bone; Otic Q. = Otic process
of the quadrate bone; Pal. = Palatine bone; Para. = Parasphenoid bone; Pter. - Pterygoid
bone; 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, seen
from the left. B. Corresponding region of a paralyzed 19-day chick embryo. Note the
fusion of Meckel's cartilage with the articular process of the quadrate. The retroarticular process of the mandible is smaller in the paralyzed embryo, and lacks the
upward angulation. Drawings prepared by tracing from photographs of KOHAlizarin-glycerol preparations.
Development of joints
353
(b) Articulations of the cervical spine. Serial sections were made of the necks
of five paralyzed embryos, three in the sagittal plane and two in the transverse
plane. Each specimen included three to ten vertebrae. A series of normal
embryos from 14 to 19 days of incubation age were also studied.
In the normal chick, the mobile joints of the neck include those between the
atlas, axis and occiput, at which gliding and/or pivoting motions occur; those
between the vertebral bodies (intercentral), which permit gliding and hinge
action; and those between the vertebral articular processes (interneural), which
Mand.
Q.J.-J.-M.
Para.
Pter.
Orb. Q.
Artie. Q.
Q-J.
Otic Q.
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-day
embryo, 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 these
joints contain well-formed cavities. In the paralyzed embryos, cavities were
invariably absent at the articulations of the vertebral bodies, the atlas, axis
and skull. These structures were solidly fused from end to end (Fig. 4A).
Small clefts or interrupted lines, which were often seen between adjacent
vertebral centra, presumably represented the sites of the original interzones.
The 'interneural' articulations were ill-defined, since the pre- and especially
post-zygapophyseal processes were poorly developed. In some specimens, the
postzygapophyses were absent. However, small articular areas were found
along the zones of contact of successive neural arches. These articulations
23
J E E M 22
354
P. D. F. MURRAY AND D. B. DRACHMAN
Orb.Q.
Development of joints
355
were undoubtedly functionless, because of the rigid fusions of the vertebral
bodies. Some, but not all of the contiguous neural arches were fused.
(c) Larynx and Trachea. The larynx and trachea were studied in serial
sections of three 18-day normal embryos, and five botulinum-treated specimens.
In the normal embryos, diarthrodial joints were present at three sites in the
larynx: (1) between the ventral process of the arytenoid and the lateral component of the cricoid; (2) between the dorsal and lateral components of the
cricoid and (3) between the ventral component of the arytenoid and the dorsal
component of the cricoid. Well-formed articular cavities were found at the
first of these joints in all the embryos studied, while cavitation was variable
at the other locations.
In the paralyzed specimens, none of the articulations had patent cavities.
Fibrous or cartilaginous fusion was invariably present, sometimes obscuring
the 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. = Inner
condyleof 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 the
mandible; Odont. = Odontoid process of the axis vertebra; Orb. Q. - Orbital process of
the quadrate bone;Para. = Parasphenoid bone; Pter. = Pterygoid bone; Q.J. = Quadratojugal bone; Sa. = surangular bone; Sa. Adv. Cart. = Adventitious cartilage of surangular
bone.
Fig. 3 A. Part of a coronal section through the quadrate and related bones of the
skull 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's
cartilage and the quadratojugal. Neither the orbital process of the quadrate nor
the medial process of Meckel's lies in contact with the parasphenoid bone. The
surangular bone has well-developed adventitious cartilage. The open spaces within
the 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 area
comparable to that shown in A. Note the absence of an articular cavity and the
fusion of the articular process of the quadrate and Meckel's cartilage. The orbital
process 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 and
parasphenoid bones. The surangular bone lacks adventitious cartilage.
Fig. 3C. A coronal section from a paralyzed 19-day-old embryo through the
quadrate and related bones. This section shows striking distortion of the quadrate
bone with marked widening of its intercondylar region. The medial process of
Meckel's cartilage is brought into abnormal contact with the parasphenoid. This
section 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) articulating with the quadrate. Note the absence of adventitious cartilage which should
normally be present on the quadratojugal.
Fig. 3E. From a paralyzed 19-day-old embryo. The cartilaginous head of the otic
process articulating with the bony squamosal. Adventitious cartilage, which
should normally be present on the squamosal is absent. There is no joint cavity.
23-2
356
P. D. F. MURRAY AND D. B. DRACHMAN
Development of joints
357
are so close together, they inevitably move upon one another. A series of
bursae, 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 were
joined by fibrous connective tissue or cartilage (Fig. 4F, G).
///. Fusion
In the normal embryos, fusion never occurred at any of the mobile articulations
of the skull, cervical spine, larynx or trachea. Points of contact between articulating skeletal elements were separated by joint cavities and/or other specialized
articular structures. In the paralyzed embryos, fusion regularly took place
across the presumptive joint regions, and at other sites where skeletal elements
were in contact. The fusing tissue was cartilaginous, bony or fibrous, depending
in part on the composition of the elements being fused. Many examples of each
type of fusion were present in the material from the botulinum-treated embryos.
(a) When the contiguous elements were both cartilages, they were fused by
cartilage or occasionally by fibrous connective tissue. In our material, such
cartilage-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 dorsaUy
in 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, making
a 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 paralyzed
19-day-old embryo. The centra are fused by cartilage. Hypapophyses are absent.
Fig. 4B. A coronal section from a paralyzed embryo, showing the pterygoids fused
to the parasphenoid, without an articular cavity or adventitious cartilage.
Fig. 4C. A transverse section through the atlas vertebra of a normal 18-day
embryo. 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-old
embryo. 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 chick
embryo, showing two mesothelial-lined bursae lying between three adjacent tracheal
rings.
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 and
ventral walls are brought artificially close together to save space. Dorsally, the
rings are fused with one another, but the continuous cartilaginous rod which
results shows periodic thickenings which must represent the positions of originally
separate rings. Fusion is not seen ventrally.
Fig. 4G. Sagittal section through the anterior part of the trachea of a paralyzed
embryo. In the roof (left) of the trachea the rings are completely fused to form
a continuous rod, without even the periodic thickenings seen in Fig. F. Unfused
rings are apparent ventrally, but no bursae are present.
358
P. D. F. MURRAY AND D. B. DRACHMAN
projected on each side, like ribs from a backbone. The trachea never became
enclosed in a cartilaginous tunnel as it would have been if all the rings had
fused completely. The cartilages of the larynx were fused by cartilage, or
occasionally by connective tissue. In some cases the cartilaginous fusion was
so complete as to obscure the site of the original articulations.
Another instance of cartilaginous fusion of two 'permanent' cartilages
occurred 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 other
cartilages with respect to fusion. In the heads of the experimental embryos,
cartilaginous fusion occurred at the quadrato-mandibular and quadrato-prootic joints (Fig. 3B, C). The skeletal elements which participate in these
articulations (i.e. the quadrate and pro-otic bones, and the Meckel's cartilage
portion of the mandible) are replacement bones. In the cervical spine, the
vertebral bodies, which are also replacement bones, were fused by cartilage
(Fig. 4A). The atlas and axis vertebrae were joined by cartilaginous bridges
ventrally, and the odontoid process of the axis was fused to the floor of the
atlas by cartilage (Fig. 4C, D).
At the site of these fused articulations between replacement bones, cartilage
extended across the presumptive joint regions, establishing continuity between
the opposed elements. Sections often showed traces of the original joint interzones in the form of lines or small clefts partially breaking the continuity of
the cartilage. In some specimens, loose connective tissue occupied the regions
adjacent to the cartilage bridges.
(b) When the contiguous elements were both bones, they were fused by bone
or 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 the
paralyzed embryos, membrane bones failed to develop 'secondary' cartilages
on their articular surfaces (see p. 359). At articulations between two membrane
bones, the bare bone surfaces faced each other directly. Such articulations were
fused by bone or fibrous connective tissue in the botulinum-treated embryos.
Thus, bony fusion was present in three of the four sectioned specimens at the
pterygo-parasphenoid joint (Fig. 4B), while the fourth was fused at a point
posterior to the usual site of articulation. The pterygo-palatine joint was
fused by bone in one case, and by connective tissue in two of the three other
sectioned 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, the
ventral portions of the same vertebrae, which originate as replacement bones,
were fused by cartilage.
In the paralyzed embryos, distortions of certain skeletal elements resulted
Development of joints
359
in the apposition of parts of bones which do not normally make contact. Bony
or fibrous fusion took place at these extra-articular sites. Thus, the articular
and orbital processes of the quadrate, and the pterygoid, were fused with the
parasphenoid bone in many of the experimental embryos (Fig. 3B, C).
(c) When one of the contiguous elements was cartilage and the other was
bone, fusion took place by fibrous connective tissue. Three examples of fusion
of heterogeneous skeletal elements were found in the experimental material,
i.e. at the articulations between the quadrate (a replacement bone), and the
quadratojugal, pterygoid and squamosal bones (which are all dermal bones)
(Fig. 3B-E).
It was particularly striking that the quadrato-squamosal joint was always
fused by fibrous tissue, while the immediately adjacent articulation between
the quadrate and the pro-otic (a replacement bone) was fused by cartilage.
There are normally two small articulations between the quadrate and the
pterygoid, and a narrow duct which passes between the pneumatic spaces in
the two bones at a site distinct from either joint. In the paralyzed embryos
the only remnant of the two articulations was a fibrous band connecting the
end of the pterygoid to the quadrate. However, fibrous connective tissue and
occasionally bony trabeculae surrounded the pneumatic duct, and helped to
anchor the two bones together. This periductal fusion falls into the group
described above for extra-articular sites.
IV. Secondary cartilage formation
In the normal chick, 'adventitious' cartilages were present at the articular
surfaces of membrane bones, in the eight locations previously described by
Murray (1963). In the paralyzed embryos, adventitious cartilage was invariably
absent (Fig. 3A-E). Furthermore, the late-forming 'articular' cartilage pads
normally found at the pterygo-parasphenoid joint were missing.
In contrast, the primary cartilage of replacement bones, trachea and larynx
was present and histologically normal in the experimental embryos, indicating
that the botulinum toxin treatment did not directly inhibit chondrogenesis.
V. Distortions of the skeleton
Although the general appearance of the head and neck was remarkably
natural in the experimental embryos, the skeletal elements were distorted in
important details. This section contains a descriptive account of examples of
such distortions which occurred consistently in the experimental material.
(a) Mandible. In all of the paralyzed embryos the tip of the mandible
protruded beyond the upper beak, by an average of 1-9 mm (Fig. 5). In order
to determine whether the upper beak was abnormally short or the lower was
abnormally long, several measurements in paralyzed and 19-day-old normal
embryos were made: (1) from the tip of each beak to the 'gape' or opening
of the jaws; (2) from the tip of each beak to the ext. auditory meatus; (3) from
360
P. D. F. MURRAY AND D. B. DRACHMAN
the tip of the upper beak to the posterior border of the skull; (4) from the tip
of 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 paralyzed
than in the normal embryos. Possible explanations of these findings are
presented in the discussion.
I
mm
Fig. 5. The head of a paralyzed embryo, to show the underhang of the mandible.
Table 1. Average measurements of beak length
Distances in mm
A
/
19-day
normal*
Underhang
Upper beak length
Mandibular length
Retroarticular process of mandible
Tip to 'Gape' upper
lower
Tip to EAM upper
lower
T\'(f
L/inerence
Botulinum(Botulinum
treatedj
minus normal)
0
25-64
19-65
2-82
1-2
119
2-33
2-30
1-9
24-54
20-31
2-57
109
1-24
2-2
2-38
1-9
-01
0-66
-0-25
-011
005
-013
008
* 5 specimens. t 8 specimens
(b) MeckeVs cartilage. The retroarticular process of Meckel's cartilage
extended straight backwards in the experimental embryos, rather than being
angled upwards, as in the normal. It was both shorter and slighter than in the
normal (Fig. 1 A, B).
In normal embryos, the medial process of Meckel's cartilage did not come
into contact with the floor of the cranium. In the paralyzed specimens, the tip
of the medial process of Meckel's was sunken into deep or shallow indentations
in the parasphenoid bone in every specimen (Figs. 2B; 3C).
Development of joints
361
(c) Quadrate. In normal embryos, the quadrate bears two accessory structures
known 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 thought
to prevent backwards dislocation at the joint. In the paralyzed embryos both
of these accessory structures were absent, although the posterior end of the
quadratojugal occupied approximately its normal position.
The orbital process of the quadrate made abnormal contact with the parasphenoid bone at the base of the cranium, which it indented and fused with
in many cases (Fig. 3B, C).
(d) Cervical spine. In the paralyzed embryos, the cervical spine was bent
1 cm
0-5 cm
10
Rostral
Caudal
Fig. 6. Drawings prepared by tracing from photographs. A. Thefirsteleven vertebrae
of 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 in
which ossification has not yet replaced the cartilage. The vertebral column is neither
bent nor twisted. Stippled areas represent the vertebral centra seen from dorsally
through the gaps between the neural arches. Neural arches three and four are
pierced by foramina for the dorsal divisions of spinal nerves. Notice the narrow
neural arches; compare Fig. 6B. Numerals indicate the serial numbers of vertebrae.
B. The first ten vertebrae of the neck of a paralyzed embryo, in approximately
dorsal view. Because of the torsion of the specimen, its rostral and caudal parts
were photographed separately, after arrangement of each part to bring its dorsal
surface into view. In the rostral portion (left), vertebrae one to three are seen somewhat 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. The
line of demarcation between the fused atlas and axis is indicated by a dotted line, in
the position of an apparent seam where the two neural arches join. The mid-dorsal
gap in the neural arch of the atlas, and similar gaps in the axis, indicate areas of
incomplete ossification. In vertebrae three, four, five and ten similar mid-dorsal
lines of incomplete ossification are indicated. No attempt is made to show the ventral
parts of the vertebrae because of the limited size of the mid-dorsal intervertebral
apertures, and because the fusion of all centra makes the floor of the spinal canal
featureless.
362
P. D. F. MURRAY AND D. B. DRACHMAN
laterally, and rotated about its long axis. The bend was either to the right or
to the left, and the rolation was either clockwise or counter-clockwise. (Fig. 6B).
In the normal embryos hypapophyses were present on vertebrae three to
eight. These were of two sorts: on vertebrae three to five, they were mid-ventral
cartilaginous protrusions from about the middle of the length of each vertebra,
piercing through the perichondral bone. In vertebrae six to eight they arose
from the epiphyseal cartilages of the vertebrae close to their anterior ends.
The hypapophyses served as attachments for the cervical musculature. In the
paralyzed 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 and
other 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 squamosal
to the retro-articular process of Meckel's cartilage and mandibular bones.
External adductor of mandible (m. adductor mandibulae externus) from the
squamosal to the mandible.
Internal adductor of the mandible (m. adductor mandibulae internus) from
the palatine to the medial process of Meckel's cartilage and its shaft.
External pterygoid (m. pterygoideus externus) from the pterygoid to the
mandible.
Quadrate-mandibular, or middle adductor of the mandible (m. adductor
mandibulae medius) from the orbital process of the quadrate to Meckel's
cartilage and the surangular bone.
Grossly, the muscles were extremely atrophic and loose in texture, with fatty
infiltration. However, all of the muscles but one were identifiable, and could
be traced from their normal origins to their normal insertions. The orbitoquadrate (m. spheno-pterygo-quadratus) which normally extends from the
orbital region near the mid-line to the quadrate and pterygoid, could not be found
in the paralyzed embryos.
Tendons, and ligaments closely associated with muscles, were more tenuous
than in normal embryos, or were absent.
Historically, the most striking finding was the devastating reduction in
muscle bulk, with the greatest part of the muscle replaced by adipose tissue.
The remaining muscle fibres were atrophic, degenerating or myotubal. In these
fibres, there was an Apparent) increase in the number of sarcolemmal nuclei,
with alignment, clumping and pyknosis of nuclei. Some of the muscle fibres
retained their striations while undergoing atrophy; others showed marked
degeneration, characterized by eosinophilia, swelling, and floccular changes,
Development of joints
363
with phagocytosis by histiocytes. In transverse section, some normal-sized
fibres had the characteristic appearance of myotubes with either a space or
a nucleus in the centre of a ring of myofibrils. The effects on skeletal muscle of
prolonged treatment with botulinum toxin have been discussed in detail
elsewhere (Drachman, 1968) and are believed to be the result of 'pharmacological denervation' caused by impairment of neuromuscular transmission.
DISCUSSION
Contractions of skeletal muscle begin early in development, and assume
considerable prominence during embryonic life, in the many species where
they have been studied (Angulo y Gonzales, 1935; Coghill, 1934; Kuo, 1938;
Minkowski, 1920; Tuge, 1931; Windle & Griffin, 1931). In the chick, somatic
movements begin at 3^- days of incubation, and increase thereafter in frequency
and duration, until, at 13 days they occupy 80 % of the embryo's time (Hamburger & Balaban, 1963; Hamburger, Balaban, Oppenheim & Wenger, 1965).
That these movements play an important role in the development of articular
and 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 paralyzing
agents in order to define the precise role of movement in the development of
simple types of joints and 'adventitious' cartilages (Murray & Smiles, 1965;
Drachman & Sokoloff, 1966). These studies indicated that skeletal muscle
contractions are essential during embryonic development for the initial formation
of joint cavities, development of 'adventitious' cartilages and certain sesamoid
cartilages, and prevention of ankylosis of joints. The present study was undertaken to investigate the effect of more prolonged paralysis on a wider variety
of articular and skeletal structures. For this purpose, immobilization of chick
embryos was maintained from the 7th through the 19th days of incubation by
means of botulinum toxin, which proved to be the most effective and convenient paralysing agent.
Type A botulinum toxin is a pure cry stall izable protein, produced by a strain
of the anaerobic bacterium, Clostridium botulinum. It causes long-lasting
paralysis 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 gas
exchange across the chorioallantoic membrane, up to the 20th day of incubation
(Romanoff, 1960) it readily survives large doses of botulinum toxin or other
neuromuscular blocking agents.
Apart from muscular atrophy, which has been discussed in detail elsewhere
(Drachman, 1968), the abnormalities in the botulinum-treated embryos were
confined to the skeletal system and related integument. The effects of botulinum
364
P. D. F. MURRAY AND D. B. DRACHMAN
toxin on skeletal and articular structures, like those of other neuromuscular
blocking agents, have been shown to be due to paralysis per se, rather than to
some unrelated toxic; action (Drachman & Sokoloff, 1966). The outstanding
changes in the present material included: (1) absence of joint cavities; (2)
fusion of joints and some non-jointed structures; (3) absence of adventitious
and 'articular' cartilages; (4) skeletal distortions.
These abnormalities are most simply explained on the basis of lack of
mechanical activity of muscle.
(1) The absence of joint cavities
In paralyzed embryos, nearly all of the articular cavities which are normally
present in the head and neck were absent. This general rule held true regardless
of 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 neck
lacked cavities. Even the diarthrodial articulations between the laryngeal
cartilages and the bursal sacs between the tracheal rings were without joint
spaces. In only three of the many joints studied were well-formed cavities
present. Incomplete joint clefts were also seen along the lines of fusion of
adjacent vertebral centra.
These observations confirm and extend previous published reports in which
immobilization of the relatively simple joints of the lower limbs of chick
embryos 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 diarthrodial
joints irrespective of their normal mechanical action, or of the origin and
composition 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. Three
possible 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 the
joints, leading to cavitation; (c) maturing synovial cells may possibly differentiate
in the absence of movement, and form small or partial clefts. The normal role
of movement appears to be one of rupturing the thinned mesenchymal tissue
between these tiny spaces, thus permitting them to become confluent.
(2) Fusion
The opposing articular surfaces of paralyzed joints were bound together
by fibrous tissue, cartilage or bone. Which of these tissues fused a given joint
depended in part on whether the articulating surfaces were composed of cartilage
or bone. Any two opposing surfaces might be bound by fibrous connective
tissue. However, cartilaginous fusion occurred only between two cartilages
and bony fusion only between two bones (Fig. 7). Thus, for example, permanently
Development of joints
365
cartilaginous structures such as the larynx and trachea were more or less
firmly fused by either cartilage or fibrous tissue. Similarly, articulations between
two temporarily cartilaginous (replacement) bones were found to be fused
by either cartilage or fibrous tissue. Examples of this type of fusion were
found at the articulations between replacement bones such as the quadrate
and Meckel's cartilage, the quadrate and the pro-otic, and in the neck, between
the vertebral centra.
Cartilage
F
Bone
C or F
B or F
Cartilage
Bone
Fig. 7. Diagram to illustrate type of tissue which fuses various combinations
of cartilage and bone. B = Bone; C = Cartilage; F = Fibrous Connective tissue.
Articulations between two bones not covered by articular cartilage were
joined either by bony trabeculae, or more often by fibrous tissue. Since articular
cartilages were missing from membrane bones in the paralyzed embryos (see
p. 359), joints between membrane bones such as the pterygoid and parasphenoid
were fused in this fashion. Joints between one bony and one cartilaginous
surface were almost invariably bound by fibrous connective tissue. Finally,
fibrous or bony fusion occasionally developed in the paralyzed specimens
between adjacent bones which do not normally articulate, such as the quadrate
and parasphenoid.
From these findings it appears that fibrous connective tissue can form between
immobilized articular surfaces regardless of their cellular composition. By
contrast, the formation of cartilage or bone in the joint interspace requires
two opposing surfaces of the same substance, suggesting that these more
specialized tissues must be derived in some way from the articular surfaces.
In a previous study, it was found that fibrous ankylosis takes place earlier than
cartilaginous fusion in paralyzed limb joints (Drachman & Sokoloff, 1966). It is
likely 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 articular
elements.
The question of whether fusion is 'primary' or 'secondary' actually becomes
a semantic one. 'Primary fusion' is usually taken to mean failure of a joint
to separate in the first place (non-fission), while 'secondary fusion' refers to
fusion of skeletal elements which were previously separate. If cavity formation
is used as the criterion of separation of elements, then the immobile joints,
which do not develop cavities, may be said to undergo 'primary fusion'.
366
P. D. F. MURRAY AND D. B. DRACHMAN
Nevertheless, the cartilaginous or bony union supervenes between two elements
which 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 of
dermal bones. It appears rather late in embryonic development, about the
1 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 movement
is necessary for the development and continued presence of adventitious
cartilage. Embryonic joints which were immobilized by grafting or pharmacological paralysis before adventitious cartilage had appeared did not develop
it throughout the limited age range studied. When immobilization was begun
after initial formation of adventitious cartilage, it was gradually replaced and
covered over by bone. Finally, artificial mechanical stimulation led to development of adventitious cartilage on the quadratojugal.
In the present study, long-lasting paralysis which was begun early and
maintained throughout embryonic life resulted in permanent absence of
adventitious cartilage. This amply confirmed the principle that cartilage
development in this situation is dependent on mechanical forces normally
provided by skeletal muscle activity.
Another similar joint structure, 'articular cartilage', was also absent in the
experimental chick embryos. Articular cartilage is normally found on both
sides of the joint between the pterygoid and parasphenoid bones, and has been
distinguished from adventitious cartilage by its location and histology. It is
located in the fibrous layers of the periosteum, separated from the bone by
soft tissue, and is comprised of typical hyaline cartilage, rather than the hypertrophic form which characterizes adventitious cartilages. The fact that 'articular
cartilage' behaved like adventitious cartilage in the paralyzed embryos suggests
that its separate classification on the basis of histological criteria may be
artificial; it may be more meaningful to group it with other 'adventitious
cartilages' 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 metabolism
of chondrogenic cells. It is clear from this and previous work that immobilization
produced by a variety of methods does not prevent all cartilage formation; the
primary cartilage models of the chondrocranium, vertebrae and limbs, and the
permanent cartilages of the trachea and larynx develop independently of
mechanical action.
(4) Distortions of the skeleton
In broad outline, the skeleton of the head and neck was remarkably natural
in the experimental embryos. However, in many important details, the movable
Development
of joints
2>61
part of the skeleton was found to be distorted. These distortions can be grouped
under^several headings:
(a) Accessory joint structures. Murray (1963) has described a number of
knobs and protrusions adjacent to certain of the cranial articulations of the
chick, which normally serve to guide and limit the motion of the articulating
bones. In the paralyzed specimens, such accessory joint structures as the
'stop' and 'condyle' were absent. While the precise mode of their formation
has'not been ascertained, it appears that they depend for their development on
muscular activity.
(b) Prominences to which muscles attach. Several skeletal prominences which
normally give attachment to muscles were either missing or grossly distorted
in the experimental embryos. For example, the hypapophyses of the vertebrae,
processes to which the cervical muscles attach, were entirely absent in the
paralyzed specimens. Similarly the retroarticular process of Meckel's cartilage,
which normally curves upwards, in line with the pull of the mandibular depressor
muscle, extended straight backwards in the paralyzed embryos. Distortions
such as these are undoubtedly due to the lack of the molding influence which
repeated muscular pull normally exerts on skeletal structures.
(c) Malposition of skeletal elements. In many respects the anatomical relationships between skeletal elements were distorted in the paralyzed embryos. Some
of these distortions were attributable to deformities of the bones themselves,
due to one or more of the factors outlined above. Additional mechanisms
which account for certain skeletal malpositions include:
(1) Fixed retention of embryonic postures. Normally, the neck of the chick
embryo 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 the
flexibility of the intervertebral joints. However, in the paralyzed embryos, the
embryonic posture is retained, due to fixation of the joints. The vertebral
column of the paralyzed embryos may acquire unnatural twists, due to the
pressures imposed by growth within a confined space, unopposed by muscular
activity (Sullivan, 1966).
(2) Lack of padding, due to muscle atrophy. In certain regions the skeletal
muscles' bulk is normally interposed between cranial bones, and serves as
a buffer to keep them apart. For example, the space between the quadrate
bone 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, these
muscles were very atrophic, permitting the quadrate bone to come into contact
with the orbital wall. The lack of muscle bulk in this situation accounted for the
narrowness of the anterior skull in the experimental embryos.
(d) Protrusion of the mandible. In the paralyzed embryos, the lower beak
protruded nearly 2 mm beyond the upper, whereas in normal chicks the beaks
meet at their tips. Theoretically the appearance of a protruding mandible
might be caused by: (a) an abnormally long mandible; (b) a short upper beak
368
P. D. F. MURRAY AND D. B. DRACHMAN
or (c) malposition of the skeletal elements favoring a forward position of the
mandible. Measurements made in this study (see results) suggest that all three
factors contributed to the protrusion of the mandible in the paralyzed embryos.
However, the mechanism by which a single influence, i.e. lack of muscular
contraction, produced both elongation of the mandible and shortening of the
upper beak, is not at all clear. It is of interest that mice and humans with
deficiency of skeletal musculature during embryonic development showed
marked retrusion of the mandible, the reverse of the situation in chicks. Perhaps
this 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 of
articular and skeletal structures, chick embryos were kept paralyzed from the
7th through the 19th days of incubation by repeated injections of botulinum
toxin. The joints, cartilages, bones and soft tissues of the head, neck, larynx
and trachea were studied by dissection and histological studies in 19-day-old
paralyzed 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 also
occurred between tracheal rings, at syndesmotic joints and at other sites where
skeletal elements were in contact.
3. Absence of adventitious cartilage, normally found on membrane bones
at articulations.
4. Distortions of cartilaginous and bony structures. These distortions were
attributable to (a) failure of skeletal muscles to exert their normal molding
influences on these structures and (b) fixed retention of certain malpositions
resulting from growth of the embryo within a confined space, unopposed by
muscular activity.
5. Marked atrophy of the skeletal musculature and associated connective
tissue structures such as tendons and ligaments.
It is concluded that the normal development and maintenance of mobile
joints and adventitious cartilages, and the development of some features of
gross skeletal form are dependent on an active musculature.
Development of joints
369
RESUME
Le role du mouvement dans le developpement des articulations et des
structures annexes: la tete et le cou de Vembryon de poulet
Pour etudier le role des contractions musculaires dans le developpement des
structures articulaires et squelettiques, on a maintenu des embryons de poulet
paralyses du 7e au 19e jour d'incubation par des injections repetees de toxine
botulique. 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 coupes
microscopiques, 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. Des
fusions se sont egalement produites entre anneaux tracheens, aux articulations
syndesmotiques et en d'autres points ou des elements squelettiques etaient en
contact.
3. Absence de cartilage adventice, present normalement sur les os de membrane aux articulations.
4. Distorsions des structures cartilagineuses et osseuses. Ces distorsions
etaient attribuables au fait que (a) les muscles squelettiques n'exercaient pas
leur influence modelante normale sur ces structures, et que (b) certaines positions
anormales etaient fixees et conservees, provenant de la croissance de l'embryon
dans un espace reduit, et de l'absence d'activite musculaire pour s'y opposer.
5. Atrophie marquee de la musculature squelettique et des structures conjonctives associees, telles que tendons et ligaments.
On en conclut que le developpement normal et le maintien d'articulations
mobiles et des cartilages adventices, et le developpement de quelques caracteres
de la forme generate du squelette, dependent d'une musculature active.
This work was supported in part by grants from the Institute of Child Health and
Human Development, the N.S.W. State Cancer Council and the Australian Research Grants
Committee.
It is a pleasure to thank Dr B. K. Hall who, besides making the measurements quoted in
the text, also read the manuscript and made many helpful suggestions, Miss Helen Macindoe
who cut the sections and did much of the photographic work, Mr W. Webster of the Photographic Department, and Mr Michael Webb of the Department of Zoology of the University
of New England, and Mr Alfonse Giglio of the Tufts-New England Medical Center for
further photographic assistance. Mr Robert Ullrich provided expert assistance with the
drawings.
Note. This study was interrupted by the sudden and tragic death of Dr Murray in the Spring
of 1967. The help which was generously given by Mrs. Jascha Murray and by Dr B. K. Hall
has made possible the completion of the manuscript. It is my hope that this effort may
symbolize the respect and affection of all those who knew Dr Murray. D.B.D.
24
JEEM 22
370
P. D. F. MURRAY AND D. B. DRACHMAN
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(Manuscript received 7 January 1969)
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