AMER. ZOOL., 15:329-350 (1975).

Evolutionary Consequences of Skeletal Differentiation

BRIAN K. HALL

Department ofBiology, Life Sciences Centre, Dalhousie University, Halifax, N.S., Canada

SYNOPSIS. Some aspects of the differentiation, growth, and morphogenesis of the tissues
within the skeleton are discussed and related to the evolution of the vertebrate skeleton. The
tissues considered are bone, cartilage, dentine, and enamel.

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The histology of the skeletal tissues of the Ordovician agnatha is reviewed with the
conclusion that the skeletal tissues of the first vertebrates were as diverse and as specialized as
are those of present-day vertebrates. Phylogenies of skeletal tissues cannot be established.
The trend during evolution appears to have been toward reduction in amount of skeletal
tissue and in the number of types of tissues present.
The factors which determine when and where a skeletal element develops ontogenetically
are reviewed and used to discuss the origin and evolution of jaws, the evolution of mem-
brane bones and the origin of such structures as sesamoid bones. Special importance is
attached to epithelial-mesenchymal interactions.
The factors which determine what particular skeletal tissue will form at a particular site
within the body are reviewed with especial reference to modulation, germ layer derivation,
and the role of epigenetic factors.
The factors which determine size and shape of the skeleton, both ontogenetically and
phylogenetically, are reviewed and the directive role of adjacent tissues emphasized.

INTRODUCTION biologist) to contribute the sort of papers

This paper will discuss the limitations
placed on the skeleton during its evolution
by examining knowledge of the way in
which the tissues of the skeletons of present
day vertebrates and invertebrates develop,
differentiate, and react to environmental
stimuli.
This problem could be examined from
the vantage point of the evolutionary
biologist or palaeontologist who would use
the fossil record as a baseline for under-
standing the skeletal tissues of present day
species. Alternatively, it could be studied
from the standpoint of the developmental
biologist or "skeletal biologist" who could
project the results of experimental analysis
back in time so as to put the flesh of cause
and effect on the bones of the fossil record.
The express request of the organizers of
the symposium to "get participants (in this
case a developmental biologist-skeletal
The author's original research has been supported
by National Research Council of Canada grant A-5056
and by the Dalhousie University Research and De-
velopment Fund. The manuscript was critically read
by Dr. E. T. Garside and by Dr. P. Person.
329
they do not usually write but can write,"
coupled with my own background, bias,
and inclinations, dictates that I be backward
rather than forward looking.
I am going to be primarily concerned not
with the skeleton as an organ system, nor
with the elements of this organ system as
individual organs, but with the dynamics of
the cells and tissues which make up the
skeleton. In order to be able to make mean-
ingful projections as to the evolutionary
consequences resulting from the limita-
tions (or potentials) of skeletal develop-
ment and differentiation the following
points must hold true:
1) Recent and fossil skeletal tissues are
equivalent tissues produced by equivalent
cells.
2) The basic plasticity (and perhaps
function) of these tissues has remained the
same during evolution.
3) The methods of forming these tissues
(the developmental processes) have not
substantially altered over geological time.
4) The chemical composition of these
tissues (the lowest evidence of gene expres-
sion preserved in the fossil record) has not
330 BRIAN K. HALL

markedly changed with time. The possibility of correlating recent and

If these hold true we may justifiably fossil skeletal tissues has recently been
begin to examine experimental results per- greatly enhanced, because fossil skeletal tis-
taining to skeletal differentiation and plas- sues can now be decalcified and sectioned,
ticity and apply these to interpretations of both for the light and for the electron mi-
the evolution of the vertebrate skeleton. croscope (0rvig, 1951, 1957, 1965; Enlow
and Brown, 1956; Moss, 19616; Halstead,
TYPES OF SKELETAL TISSUES 19696) and because chemical analyses can
now be carried out on both the inorganic

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I should perhaps define the skeletal tis- and the organic components of these tis-
sues which will be discussed, for I shall be sues (Mathews, 1966, 1967; Ho, 1967; Biltz
concerned primarily with the hard and Pellegrino, 1969; Kobayashi, 1971;
(mineralized) tissues of the skeleton. Other Hancox, 1972). The latter studies have in-
tissues of the musculo-skeletal system, such dicated essential similarity in the chemistry
as ligaments, tendons, fibrous and other of the skeletal tissues of fossil and recent
connective tissues, vascular, muscular, and vertebrates.
hemopoietic elements, will take second
place in the discussion. This will be done, SKELETAL TISSUES OF ORDOVICI AN VERTEBRATES
not because these other tissues are unim-
portant, for they obviously are important, A brief review of the skeletal tissues of
especially in the context of the evolution of the early vertebrates will serve to illustrate
complex, multi-tissue functional units (see their homology with the tissues of the
Bock and von Wahlert, 1965; Moss, 1968<2; present-day vertebrates, their general
Alexander, 1975), but because they are not complexity and high degree of specializa-
usually preserved in the fossil record. tion, and their approximate times of ap-
The mineralized skeletal tissues may be pearance in the fossil record. I will consider
broadly classified into four types (see 0rvig, the members of the Class Agnatha (the jaw-
1967, for a detailed classification). less vertebrates), the earliest vertebrates for
1) Bone: intramembraneous and en- which fossil skeletal material is available.
dochondral, cellular and acellular, cancel- The major orders within the Agnatha and
lous and lamellar (see Smith, 1947, for an their distribution within the geological time
excellent summary of the classification of scale are summarized in Table 1. All except
bone). the Cyclostomata are extinct. The members
2) Cartilage: hyaline, elastic, fibrocarti- of the extinct orders were all small (most 50
lage, calcified, chondroid. to 300 mm in length, some Osteostraci at-
3) Dentine: cellular and acellular. taining 1 m in length) and are known cnly
4) Enamel. from fossilized cephalic shields.
5) Tissues intermediate between the The notochord is assumed to have been
above. present as a supporting soft skeleton in all
The basic cell type which produces these of these specimens. The presence or ab-
mineralized tissues has been termed the sence of uncalcified cartilage can be argued
Scleroblast by Moss (1964a), implying that only by inference (i) because cartilage, un-
the tissues represent a family or race of like the notochord, is not a universal diag-
tissues with close affinities one to another. nostic vertebrate tissue, and (ii) because if
Such cells may be mesodermal, ectodermal, uncalcified it is unfossilizable. Its inferred
or ectomesenchymal (from the neural presence or absence in the early verte-
crest). Thus, the osteoblast (mesodermal, ec- brates, especially whether it antedated the
tomesenchymal) produces bone; the chon- appearance of bone, as it does in ontogenet-
droblast (mesodermal, ectomesenchymal— ic endochondral ossification, has led to
ectodermal?—) produces cartilage; the numerous ingenious but speculative
Odontoblast (mesodermal) produces den- theories of vertebrate evolution, adaptive
tine; and the ameloblast (ectodermal) pro- radiation, and the origin of skeletal tissues
duces enamel. (Romer, 1942, 1963, 1964; Smith, 1947;
 SKELETAL DIFFERENTIATION AND EVOLUTION 331

TABLE 1. Classification and geological distribution of the jawless vertebrates (Agnatha).

Subphylum: Vertebrate
Class: Agnatha
Subclass: Cephalaspidomorphi (Monorhina)
Order: Osteostraci (middle ordovician to late devonian)
Anaspida (middle ordovician to late devonian)
Cyclostomata (Lower devonian to recent)
suborder: Petromyzontia
: Myxinoidei
Subclass: Pteraspidomorphi (Diplorhina)

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Order: Heterostraci (early ordovician to late devonian)
Coelolepida (middle ordovician to late devonian)

Berrill, 1955; Jarvik. 1959; Urist, 1962; (globular, calcospheritic) calcification has
Denison, 1963; Moss, 1961*, 1964a, been observed. Similar cartilage is seen in
1968o,c; 0rvig, 1967, 1968; Halstead, the mandible and palatoquadrate of mod-
1969a). ern sharks (Applegate, 1967) and has been
The skeletal tissues which have been pre- observed in homografted hyaline cartilage
served in the fossil record are summarized in man (MacConaill, 1973). Perichondral
in Table 2 along with their time of earliest bone was also present in some of the mem-
appearance. bers of this group in association with a
The oldest vertebrates known (Archodus, neurocranium presumed to have consisted
Palaeodus), species of unknown affinity of uncalcified cartilage. The Osteostraci
from the early Ordovician of Russia, pos- (Alaspis, Cephalaspis, Hemicyclaspis) had a
sessed a dermal skeleton which consisted of dermal exoskeleton of cellular bone capped
isolated denticles composed of dentine by dentine and enamel, and a pharyngeal
(Denison, 1963). The Anaspida (Anaspis, and cranial endoskeleton of endochondral
Birkenia, Pterolepis) possessed isolated der- bone (Fig. 1). Spherulitic and globular cal-
mal scales composed of acellular bone, the cified cartilage was also present. The ear-
so-called aspidin(e). The Heterostraci (As- liest jawed vertebrates, the Acanthodii
traspis, Pycnaspis, Eriptychius), from the early (middle Silurian to Permian) possessed a
and middle Ordovician, had a solid dermal similar range of tissues, except that their
armor of acellular, coarse-fibered aspidin, bone was always cellular.
covered by a layer of dentine which in some Thus, during the early Ordovician two
was in turn covered by a layer of enamel major combinations of skeletal tissues were
(enameloid) (Denison, 1963; Halstead, present within the Agnatha: acellular bone,
1969ft; 0rvig, 1951). In one species, Erip- dentine, enamel, and calcified cartilage in
tychius, cartilage exhibiting spherulitic the Heterostraci, and cellular bone, dentine,
TABLE 2. A summary of the occurrence of mineralized skeletal-tissues in the vertebratefossil record. First appearance of major
groups (classes) is also shown.

Quaternary re
Cretaceous -5
Jurassic Mammals c
0
ichoiidr

X
Triassic Birds
peril

Permian
lage
tine
mel

ha)

Pennsylvanian Reptila ,-
c •p re -a
Mississipian V re
rm

W a bo
Amphibia U
Devonian Chondrichthyes
Silurian Placoderms £
idi

V CO
Osteichthyes a. c
Ordovician Agnatha < CQ
Cambrian
Pre Cambrian
1
Note that acellular bone reappeared in teleosts.
332 BRIAN K. HALL
FIG. 1. Bone containing globular structures (chon-
drocytes?) from (A) a cephalaspid, and (B,C) an an-
tiarch. (From 0rvig, 1968.)
enamel, and calcined cartilage in the Os-
teostraci. The replacement of cartilage by
the formation of endochondral bone ap-
pears to have been a slightly later develop-
ment in the middle Ordovician Osteostraci
(Romer, 1964). With the possible exception
of aspidin whose homology with bone, and
especially with the acellular bone of the
teleosts, has been questioned by some, all of
the skeletal tissues of the Agnatha may be
readily equated with and appear to be just
as complex and "advanced" as those of the
living vertebrates.
One might expect to find many skeletal
tissues in these, the first vertebrates, which
were "missing links"—tissues intermediate
between one recognizable tissue such as
bone and another such as cartilage, or tis-
sues which were, for example, bone, but
bone that was simpler and more primitive
in structure than the bone described previ-
ously. Some intermediate tissues were in-
deed present in the Agnatha, however they
do not represent a phylogenetic trend, but
rather intermediates between two
specialized mineralized tissues. 0rvig
(1951) has made a very extensive study of
three principal types of dentine, of tissues
intermediate between dentine and bone,
and of tissues intermediate between bone
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and calcified cartilage, in the early verte-
brates. The considerable debate over
whether aspidin was dentine or bone,
partly a debate over whether aspidin was
cellular or not, is in itself good evidence for
the presence of intermediate tissues among
the early vertebrates (reviewed by
Halstead, 1969<2,6). The holostean fish,
Amia calva, possesses cellular dentine in
which the process leading to the persistence
of the odontoblasts is indistinguishable
from that leading to the persistence of os-
teocytes in bone (Moss, 19646). 0rvig's
(1965) contention that the basic distinction
between bone and dentine "is one of
scleroblast behavior rather than one of
scleroblast activity" would seem to be a
good summary of the interrelations be-
tween the two tissues. There were then less
clear cut distinctions between the skeletal
tissues of the Ordovician vertebrates than
exists between the tissues of present-day
vertebrates.
An oft-debated question is that of the
relationship between cellular and acellular
bone. Some of the teleosts possess acellular
bone, a consistently cellular bony skeleton
not having been universally attained until
the evolution of the tetrapods. It seems to
have been well established that in the evolu-
tion of the teleosts from the actinoptery-
gians, acellular bone was derived secondar-
ily from cellular bone (Kolliker, 1859; Den-
ison, 1963). The relationship of Agnathan
aspidin to cellular bone has been more
hotly debated, all possible views having
been proposed: that the two evolved inde-
pendently (0rvig, 1965; Moss, 1968a); that
aspidin evolved from cellular bone (0rvig,
1957, 1968); and/or that cellular bone
evolved from aspidin (Denison, 1963). The
whole question has been reviewed by Tarlo
(1964), 0rvig (1965), Halstead (1969a) and
 SKELETAL DIFFERENTIATION AND EVOLUTION 333

Hancox (1972). The fact that the two tissues AMPHIBIA"

appeared contemporaneously in the Or-
dovician in two distinct groups of Ag-
nathans indicates that the two must have Crossopterygii*
arisen earlier, either independently from Dipnoi* . 4 ^ . Teleosts*
less-specialized tissues or from a single
primitive skeletal tissue. ^SARCOPTERYGir ^
A further question which has been de-
bated back and forth for decades is: "which Jp Actinopterygii*

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of these skeletal tissues appeared first in the
vertebrate skeleton during its evolution?" OSTEICHTHYES*
usually reduced to a question of "which Chondrichthyes^. .^.
came first, cartilage or bone?" This consid-
erable debate was, to a large extent, fos-
tered by the application of embryological PLACODERMS*
theory to the study of skeletal evolution and Cyclostomes QstArms'
illustrates the need for caution when mak-
ing such applications. Because the verte-
brate embryo was found to have a pre- AGNATHA*
dominately cartilaginous skeleton, because
cartilage precedes bone in endochondral FIG. 3. The evolution of the lower vertebrates as-
bone formation, and because the car- suming bone (*) appeared early in evolution. (Mod-
tilaginous fishes (the Chondrichthyes) were ified from Romer, 1964.)
considered primitive, cartilage was as-
sumed to have evolved before bone and brates. Endochondral bone seems to have
phylogenies of the vertebrates were con- arisen later (first in the Middle Ordovician
structed accordingly (Fig. 2) (Romer 1942, Osteostraci, then in the Silurian acantho-
1963). However, as the data summarized in dians, and in some of the placoderms) and
Table 2 indicate, enamel, dentine, calcified to have come to prominence in those
cartilage, and dermal bone (both cellular groups from which later groups evolved
and acellular) share equal honors in terms (the rhipidistian sarcopterygians which
of first appearance in the ancient verte- gave rise to the land vertebrates, and the
actinopterygian fishes). Thus, the first ver-
AMPHIBIA* tebrate hard tissues were histologically di-
verse and specialized. A phylogeny which
takes these facts into account is shown in
DIPNOI Figure 3.
Crossopterygians* The question of the adaptive role played
by the skeletal tissues in the early verte-
brates has received considerable attention
with the assumption that the factors re-
OSTEICHTHYES sponsible for the adaptations were the same
factors as were responsible for the initial
Placoderms" -^fc- Ostracoderms* evolution of the skeletal tissues. The follow-
ing possible functions have been proposed:
^ CHONDRICHTHYES ^ Bone served a mechanical supporting func-
tion as it does today (Berrill, 1955; Schaef-
fer, 1961; Denison, 1963). The dermal
CYCLOSTOMATA skeleton served both as a reservoir and as a
permeability barrier to conserve calcium
FIG. 2. The evolution of the lower vertebrates as- and especially to conserve phosphorus.
suming bone (*) appeared late and off the main The ultimobranchial gland and, in later
evolutionary line. (Modified from Romer, 1964.) groups, the parathyroid gland aided in this
334 BRIAN K. HALL

function (Berrill, 1955; Smith, 1961; Urist,
1962, 1963, 1964; Moss, 1964a; Tarlo,
1964; McLean and Urist, 1968). This view
has been denied in general by Denison
(1963) and for acellular bone in particular
by Moss (1963). The dermal armor may
also have provided a defensive outer shield
(Denison, 1963; Romer, 1963). Cartilage
was thought to have served both for
a restriction of the types of tissues pro-
duced from many specialized tissues in
lower groups to few tissues, no more
specialized, in the higher groups. Moss
(1963, p. 339) states: "It is almost as if in an
essentially nonweight bearing skeleton
(that of fishes) no selective advantage is
found in one histological type of osseous
tissue or another."

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mechanical support and for rapid growth, The transition from the reptiles to the
especially embryonic growth and develop- mammals did involve fundamental changes
ment (Romer, 1942, 1963, 1964; Berrill, in the associations and positions of skeletal
1955; Denison, 1963). elements, e.g., the evolution of the new jaw
articulation (du Brul, 1964); the growth of
SKELETAL TISSUES DURING EVOLUTION the jaw (the sutural growth of the multi-
boned reptilian mandible vs. the apical
Enlow and Brown (1956, 1957, 1958) growth of the mammalian dentary—
have provided a very detailed comparative Sicher, 1966); the attachment of the teeth
survey of the histology of both fossil and to the jaw (ankylosed in reptiles and a syn-
recent bone, and more recently Enlow desmosis in mammals). These involved
(1969) has reviewed the bone of the reptiles changes in the developmental processes,
and Ricqles (1968, 1969, 1972) that of the but the tissues produced were fundamen-
tetrapods. No advance in the structure of tally the same as were those of the Ordovi-
bone was associated with the evolution of cian vertebrates. Thus, bone histology can-
the reptiles from the amphibia or of the not be used to identify individuals of a par-
mammals from the reptiles. Thus: ticular species and often not members of
" . . .while much structural variation of bone tis-
the same class (Enlow, 1966).
sue is found (throughout evolution), these differ- The conclusions to be drawn from an
ences usually involve variation in the arrangements examination of the histology of the bones of
of fundamental components and not major dif-
ferences in the structure of the components fossil vertebrates would appear to be as fol-
themselves" (Enlow and Brown, 1958, p. 212). lows: The skeletal tissues of the early Or-
Again (p. 220) " . . .in the history of bone tissue, a dovician vertebrates were essentially the
single evolutionary line cannot be recognized. It same as are those of present-day verte-
is not possible to trace a precise series of progres- brates, although more intermediate tissues
sive, increasingly complex developmental stages
from ancient fish to modern animals." were present then than are present now.
Again: "Anatomical specializations are not per The first mineralized vertebrate tissues
se necessarily correlated with phyletic modifica- known from the fossil record were complex
tions in the histological structure of hard tissues" and specialized (implying earlier unfos-
(0rvig, 1965, p. 554). silized and perhaps simpler (?) skeletal tis-
For example the degree of mineralization sues). Skeletal evolution in the vertebrates,
of the skeleton, as indicated by its density, at least since the Ordovician, has not in-
does not show a phylogenetic trend, but volved major changes in cell or tissue or-
indicates local adaptation to environmental ganization but rather has involved adaptive
conditions such as mechanical stress, responses of already specialized and plastic
equilibrium, buoyancy, protection, tem- tissues to new local environmental changes.
perature, conservation of energy, as has
been documented for the Ostracoderms THE BORDERLAND BETWEEN EMBRYOLOGY AND
(Schaeffer, 1961) and for the Cetacea (Felts PALAEONTOLOGY
and Spurrell, 1966). There is very consid-
erable variation in the histological structure As it can be maintained that skeletons of
of fish bone and cartilage (explosive radia- the earliest vertebrates were highly
tion) which is not seen in the Amphibia or specialized structures whose tissues were as
the Reptilia (Moss, 1964a, 1969), indicating complex (advanced) as are those of present-
 SKELETAL DIFFERENTIATION AXD EVOLUTION 335

day vertebrates, it seems reasonable to con-
clude that the genetic machinery required
to produce such tissues was established
equally early. The subsequent evolution of
the hard tissues of the skeleton did not in-
volve major, progressive changes in their
fundamental structure and so may not have
necessitated major changes in the genome.
If it can also be shown, as will be attempted
bone. The cells which condense either arise
locally in the position which the bone will
subsequently occupy, as in the formation of
the vertebrate, limbs, or elements of the
skull (Hall, 1971), or they migrate from
elsewhere in the body to the site at which
skeletogenesis is to occur, as in the migra-
tion of mesodermal cells into the lower jaw
of the chick (Jacobson and Fell, 1941), or

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later, that skeletal tissues are especially sus- the migration of the ectomesenchymal cells
ceptible to modulations from environmen- from the neural crest into the skull, mandi-
tal factors, then experimental studies on ble, or pharynx (Johnson and Listgarten,
skeletal development and differentiation 1972). The factors responsible for conden-
become extremely valuable as tools for un- sation are largely unknown, although cell
derstanding the evolution of the skeleton. adhesion may play a role (Ede, 1971; Moss,
This view has been aptly synopsized by 1972ft). In the case of the ectomesenchymal
Moss (1964a): " .. .intrinsic embryological cells of the neural crest which form the
phenomena, as modified by the extrinsic cartilage of the head, the stimulus for con-
factors of mechanical function, forms the densation results from interaction with the
basis of a meaningful discussion of the pharyngeal endoderm (Holtfreter, 1968).
evolution of skeletal tissue type."
We might study the interaction between Epithelial-mesenchymal interactions
these two bodies of knowledge by examin-
ing available information on the develop- The position of the condensed
mental processes involved in producing the mesoderm or ectomesenchyme, and the
skeleton. This we shall do by following the shape, size, and rate of growth of the con-
life history of a "typical bone," an approachdensed cells, are determined by interaction
used previously in reviewing the his- with adjacent ectoderm (epithelia), the so-
togenesis and morphogenesis of bone called epithelial-mesenchymal interactions.
(Hall, 1971). We will ask a series of ques- These interactions may involve the two-way
tions about skeletal development, present interchange between a stationary layer of
the current status of knowledge on that top- ectoderm and local mesoderm, as in the
ic, and then determine whether the infor- formation of the amniote limb, or they may
mation is of any value in understanding involve establishment of an association be-
aspects of the evolution of the skeleton. tween mobile (migrating) mesoderm
and/or ectoderm, as in tooth or middle ear
formation. Other mesodermal cells ajacent
INITIATION OF SKELETOGEXESIS to these specialized epithelia, but outside
the condensation, can form skeletal tissues,
The first question is a double-barreled but only the tissues within the condensation
one: How does the development of a bone do form skeletal tissues (reviewed by Hall,
commence, and what determines where 1971). A good example is the formation of
that bone will develop? the cartilaginous primordia of the long
bones in the limb of. the embryonic chick.
Mesenchymal condensation
Any mesodermal cell in the limb bud is
The first sign that skeletogenesis is im- capable of producing either cartilage or
minent is condensation of mesodermal (or muscle up to Hamilton-Hamburger stage
ectomesenchymal) cells to form the anlage 24 (4-'/2 days of incubation). Thereafter
(primordium) of the bone. The position of only the cells in the central condensed
the condensation within the embryo mesoderm form cartilage and these are the
cells most closely associated with the
defines the subsequent position of the bone specialized ridge of ectoderm—the apical
within the adult; the shape of the condensa- ectodermal ridge. An extra ridge grafted to
tion defines the future basic shape of the
336 BRIAN K. HALL
the limb bud will induce cells outside the
condensation to condense and to form ad-
ditional skeletal tissue, indicating that the
ectoderm influences the fate of the meso-
dermal cells. Thus, one can propose, as I and
others have done in the past (Hall, 1970,
1971), that potentially any mesenchymal
cell is capable of producing more than one
skeletal tissue (such as cartilage and bone)
and that during development, local en-
vironmental factors stabilize the genome of
cells in specific sites for skeletogenesis and
further influence what type of skeletal tis-
sue will form.
Teeth form at the junction between the
buccal epithelium and those ectomesen-
chymal cells which have migrated in from
the neural crest. Cells from both germ
layers are required before tooth formation
can be initiated. The odontoblast arises
from the ectomesenchyme and produces
dentine only when it is in contact with adja-
cent oral epithelium (Horstadius, 1950).
The ameloblast arises from the ectoderm,
produces enamel, and, in coordination with
the ectomesenchymal odontoblast, leads to
the formation of a normal tooth (Fig. 4).
It has been shown by Goedbloed (1964)
that the development of the mouth cavity,
the middle ear cavity and the external au-
ditory meatus, and their associated tissues,
involve epithelial-mesenchymal interac-
tions brought about by the movement of
epithelial borders into association with new
ECTODERM
neural tube neural crest=buccal epithelium
PHARYNGEAL
ENDODERM
odontoblast = = s ameloblast
dentine
enamel
TOOTH
FIG. 4. The major ectodermal-mesodermal interac-
tions involved in the formation of the tooth. Double
arrows indicate inductive interactions. (Modified from
Koch, 1972.)
mesenchyme. For example, the mesen-
chyme which produces the tissues of the
middle ear arises from several regions and,
depending on the nature of the associated
epithelium, produces cartilage, muscle, or
soft connective tissue. The same holds true
for the origin of the tissues of the lower jaw
in birds, except that it is the mesoderm (ec-
tomesenchyme?) which shifts in position.
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Cells destined to form cartilage, bone, or
muscle arise in three separate mesen-
chymal centers outside the mandible (each
associated with a "transitory epithelial
thickening") and then migrate into the
mandible where they differentiate (Jacob-
son and Fell, 1941).
There is then experimental evidence for
the shifting of epithelial-mesenchymal
borders during development. These shifts
are brought about either by movement of
site and tissue-specific epithelia or by
movement of tissue-specific mesenchyme.
Can we use this knowledge to understand
the initiation of new skeletal elements dur-
ing evolution? Two obvious areas to con-
sider are the origin of the jaws during
evolution of the gnathostomes and the
evolution of the mammalian jaw articula-
tion, both of which involve the shifting in
position, and modification of function, of
pre-existing skeletal elements.
In the evolution of the gnathostome ver-
tebrates from the Agnatha, the skeleton of
the first apparent gill arch was extended
anteriorly to form the skeleton of the jaws
(Fig. 5). The upper half of the arch formed
the upper jaw (the palatoquadrate); the
lower half of the arch formed the lower jaw
(the mandible). The skeleton of the arches
consists of cartilage and bone derived, in
part, from ectomesenchyme of neural crest
origin. The skeletal tissues of the lower jaw
include a cartilaginous rod (Meckel's carti-
lage), also probably of neural crest origin,
and a number of membrane bones, to be
discussed later. The simplest developmen-
tal model which would explain this
evolutionary sequence and which would be
consistent with the experimental data
summarized above would be as follows: as
the mouth enlarged, the mesenchyme of
the gill arch region extends anteriorly as
the primordium of the jaw, taking with it its
 SKELETAL DIFFERENTIATION AND EVOLUTION 337

which invest the cartilaginous rod. These

membrane bones were not present in the
gill arch and therefore had to be produced
de novo, and will be discussed below.
Before discussing the origin and de-
velopment of membrane bones, I will turn
to the evolution of the mammalian jaw ar-
ticulation. The reptilian jaw articulates
through the articular of the lower jaw and

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the quadrate of the upper jaw. In the transi-
tion to the mammals these two endochon-
dral bones migrated posteriorly to form, re-
spectively, the malleus and the incus of the
middle ear. A new jaw articulation de-
veloped between a newly formed condyle
of the now greatly expanded dentary
(which comprises the lower jaw) and the
squamosal of the skull. Ample evidence il-
lustrating the gradual migration of these
bones in the synapsid reptiles is provided
from the fossil record, and the concept of
shifting of epithelial-mesenchymal borders
would seem to provide an adequate
mechanism to explain the developmental
processes involved. The search for the
FIG. 5. The origin of the jaws, /f .jawless Agnatha.B, stimulus which initiated the movements in-
Gnathostome. First gill arch modified as jaws. C, Os- volves consideration of the skull as a func-
teichthyes. Jaws braced by second gill arch. (From du
Brul, 1964.) tional unit and the changing conformation
of the skull base, changes in muscle mass,
associated specific epithelium, and produc- new directions of stress (see below and also
ing in the new site, a cartilaginous and bony Noble, 1973). It may be that no fundamen-
rod as it would have done in the gill arch. tal changes in the genome were required to
The alternatives of establishment of a new bring about the development of this new
pathway of migration of neural crest di- articulation.
rectly from the neural tube to the anterior
segment of the lower jaw and of subsequent
establishment of new site and tissue-specific The origin of membrane bones
epithelia to induce the now adjacent
mesenchyme to produce Meckel's cartilage The question of the origin of the mem-
appears less likely (i) because several de- brane bones of the lower jaw, or indeed, of
velopmental processes are involved, and (ii) membrane bones in general, irrespective of
because the pre-existing cartilage- their location, is one to which we shall now
determined tissues within the gill arch turn. The factors which induce mesen-
could be transposed more readily. How- chymal cells to modulate to osteoblasts and
ever, in the avian embryo, neural crest cells to deposit bone matrix in vivo have not
do follow such a route along a cleavage been determined, except for those mem-
plane between the ectoderm or pharyngeal brane bones surrounding the brain and
endoderm and the adjacent mesoderm forming the dermocranium and for the
(Johnson and Listgarten, 1972). periosteal bones associated with the carti-
In addition to Meckel's cartilage and an lage models of long bones.
endochondral bone(s) which develops in The intramembraneous (perichondral,
Meckel's cartilage (see below), the lower jaw periosteal bone) which forms around the
contains a number of membrane bones shafts of endochondral bones has been
338 BRIAN K. HALL
shown to form under the influence of an
induction from the associated hypertrophic
chondrocytes (Mareel, 1967). These chon-
drocytes are in turn either replaced by, or
converted into, endochondral bone (Hol-
trop, 1966; Crelin and Koch, 1967; Hall,
1970). A similar situation has been postu-
lated for the initiation of membrane bone
formation around Meckel's cartilage, al-
though here the evidence is not as good.
Frommer and Margolies (1971), from
studies of normal development of the
mandible in the mouse, maintain that the
close spatial relationship between initial
chondrogenesis of Meckel's cartilage and
initial intramembraneous ossification in the
mandible indicates induction of membrane
bone by Meckel's cartilage. Further, they
feel that the membrane bone so induced in
turn enables the adjacent areas of Meckel's
cartilage to undergo endochondral ossifica-
tion. One could suggest that the membrane
bones surrounding Meckel's cartilage (and
this is a topographic arrangement not dup-
licated elsewhere in the body) were origi-
nally a single perichondral bone developed
in the perichondrium of the skeleton of the
gill arch. If during transformation into the
lower jaw, parts of Meckel's cartilage sub-
sequently failed to undergo hypertrophy,
subdivision of the single bone could have
occurred allowing it to loosen its previously
close connection with the cartilage. Friant
(1959, 1964, 1966, 1968, 1969) has pub-
lished an extensive series of observations
indicating that Meckel's cartilage is com-
pletely replaced by endochondral bone in
some mammalian species, incompletely re-
placed in others, and that in some it re-
mains as a cartilaginous rod, the chondro-
cytes failing to hypertrophy. In those where
replacement is partial, bone forms only ad-
jacent to hypertrophicchondrocytes. Other
areas of the cartilage fail to undergo hyper-
trophy and remain cartilaginous.
Many membrane bones do not develop in
proximity to primary cartilage. Urist (1962,
1970) maintains that these membrane
bones are induced by the adjacent fibrous
connective tissue, although experimental
evidence is lacking. The membrane bones FIG.
notochord and overlying mesenchyme responsible for
surrounding the brain develop from formation of cranial dermal bones in embryonic chick.
mesenchyme whose skeletogenic potential (From Schowing, 1968c.)
is activated by influences from the brain
and from the notochord. Of potential use
in the study of the evolution of the dermal
skeleton is the work of Schowing
(I968a,b,c.) He has shown that, in the em-
bryonic chick, the brain, nerve cord, and
notochord induce overlying mesoderm to
form the intramembraneous bones of the
skull and to form them in the appropriate
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spatial relationships to one another (Fig. 6).
The strong development of the dermal
head skeleton in the Ordovician agnathous
vertebrates indicates that this mechanism
may have been established at the outset of
vertebrate evolution. The ventral and
ventro-lateral components of the tetrapod
neurocranium, e.g., the occipital and the
basisphenoid (Fig. 6), are all endochondral
in origin, whereas the more dorsal elements
are of course intramembranous. Have we
here a mechanism for distinguishing what
tissue type a given mesodermal condensa-
tion will initially form? Could one speculate
that it is a combination of inductive
influences emanating from both the
notochord and the central nervous system
which determine endochondral ossifica-
tion, whereas neural influences alone de-
termine intramembranous ossification?
The somatic mesoderm in which the ver-
tebrae develop also requires an induction
from the notochord before chondrogenesis
can commence (Lash, 1968). As in the de-
velopment of the endochondral bones of
the skull, the endochondral vertebrae re-
ceive a dual inductive stimulus both from
the notochord and from the spinal cord. In
fact in some vertebrates, e.g., the salaman-
6. The inductive interactions between brain,
 SKELETAL DIFFERENTIATION AND EVOLUTION
ders, cartilage develops within the
notochord (Wake and Lawson, 1973).
To summarize the answer to the
double-barreled question, the site and tim-
ing of skeletogenesis depends primarily on
ectodermal-mesodermal interactions, a
concept which can be very usefully applied
to the study of the evolution of skeletal tis-
sues and of the skeleton.
TYPES OF SKELETAL TISSUES
This leads into a discussion of the second
question: What determines the type of
skeletal tissue that will develop?
Modulation and the scleroblast
It is only after the mesenchymal cells
have condensed that skeletogenesis begins.
Depending on the site within the body and
on local epigenetic factors, one of the fol-
lowing types of scleroblasts will form:
chondroblast, chondrocyte (cartilage); os-
teoblast, osteocyte (bone); fibroblast (liga-
ment, tendon); odontoblast (dentine);
ameloblast (enamel). Each is characterized
by a particular extracellular product and by
a particular structure. Along with the con-
cept that any mesenchymal cell has the po-
tential to become a scleroblast given the
appropriate stimulus is the concept that the
various types of scleroblasts are members of
an interrelated and potentially intercon-
vertible family (Moss, 1964a, 1968c, 1969;
Hall, 1970, 1971). The existence, in both
recent and fossil vertebrates, of tissues in-
termediate between two well-defined
skeletal tissues (cartilage and bone; bone
and dentine) provides the histological basis
for this concept. The conversion of one
scleroblast type into another after experi-
mental manipulation provides the empiri-
cal verification of the concept. Thus, to en-
deavor to understand why a particular
skeletal tissue develops at a particular site in
the skeleton is to ask the question: How do
different scleroblasts modulate from com-
mon stem cells and what causal factors are
involved?
Ectomesenchyme of the neural crest
The derivatives of the neural crest pro-
339
vide an illuminating example of the con-
cept that histologically quite disparate
skeletal tissues may arise from common
stem cells. The neural crest is derived from
the ectoderm at the border between the
epithelial ectoderm and the developing
neural ectoderm and separates off as the
latter transforms into the neural tube.
These cells break free, become mesen-
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chymal in appearance (hence the name ec-
tomesenchyme) and migrate to various sites
within the embryo. In the Amphibia, the
chondroblasts, osteoblasts, and odonto-
blasts of the cranial skeleton all arise from
the neural crest (de Beer, 1958). Experi-
mental studies on the chick embryo have
provided similar conclusions for the origin
of cranial chondroblasts and several exper-
iments will be discussed.
Johnson (1966) carried out a study in
which excised portions of the neural crest
of 30-hr chick embryos were replaced with
comparable pieces previously labeled with
H3-thymidine. Subsequent examination as
late as 9 days of incubation indicated
labeled chondrocytes (and therefore carti-
lage) in the cartilages of the head and in the
visceral arches. Although labeled bone was
not observed (and osteogenesis is well ad-
vanced in the head of a 9-day embryo),
labeled undifferentiated mesenchymal cells
were seen and were thought to be os-
teogenic precursors.
Hammond and Yntema (1964) removed
neural crest without replacement from
similarly aged embryos and noted sub-
sequent depletion of cartilage from the
head and lower jaw. No mention of bone
depletion was made. Thus, in the chick, the
origin of head cartilage from neural crest
rests on sound experimental grounds; ori-
gin of bone from similar cells is based on
inferential evidence (further evidence is
being sought in my laboratory).
Jollie (1971) has studied the develop-
ment of the head of the embryonic shark,
Squalus, and has shown that Meckel's carti-
lage and the teeth (both of which are de-
rived from the cells of the neural crest) arise
from cells in a common blastema situated
under the epidermis. These cells then
stream into the future positions of these
organs, where they differentiate. Thus,
340 BRIAN K. HALL

similar stem cells form different skeletal tis- both fossil and recent vertebrates, and Jol-
sues according to site-specific factors. Hall lie (1968) has provided an excellent discus-
(1970, 1971, 1972) has reviewed many of sion of the neural crest and of the conse-
the factors involved in such transforma- quences of acceptance of the Delamination
tions and an example is discussed later. Theory (to be discussed below). If the
The sharks, despite their undoubted po- neural crest of the early vertebrates pos-
sition as cartilaginous fishes, contain sessed the ability to form a variety of
mesodermal cells capable of producing skeletal tissues, as appears, based on the
bone. Bone has been described in the mod- tissues present in fossil Ordovician verte-

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ern sharks and chimeras (Peyer, 1968): at brates, to be the case, then the skeleton of
the base of the teeth in Squalus acanthias, these vertebrates was a highly adaptive or-
where it is acellular (Moss, 1970), and in gan, ideally suited to evolutionary mod-
Heptanchus (Holmgren, 1942). Bone has ification. Mute fossil bones do speak when
also been observed in Ornithoprion hertwigi, asked the right questions.
a specialized Permian edestidaed shark.
This species was partially armored, the Delamination
armor consisting of denticles and scales, the
bases of which were imbedded in bone. The previous studies provide experi-
These bony elements were fused together mental evidence for the delamination
to form a thick layer of bone around the theory proposed by Holmgren (1940) and
snout and the mandible (Zangerl, 1966). modified by Jarvik (1959) and Moss (1968c,
Whether these bones are of neural crest 1969). In this theory it is postulated that
origin is unknown. Whether they are or are dermal skeletal tissues, formed from the
not, they illustrate the hidden potential re- ectomesenchyme of the neural crest, lose
sident within skeletogenous cells, and indi- their contact with the epidermis and sink in,
cate that views such as, "the process of bone to be replaced at the surface by a succeed-
development is, so to speak, much more ing layer of skeletal tissues, not necessarily of
firmly imbedded in the embryological pat- the same type as the first-formed tissues. The
tern of these (Osteichthyes) fishes" (than in first layers to sink in by delamination form
Chondrichthyes) (Romer, 1964, p. 76), are the endoskeleton, the last-formed layers
too restrictive in concept. remain at the surface to become the dermal
exoskeleton (Tarlo, 1964; Moss, 19686).
Much of the skeleton of the early verte- Hay (1964) has shown that collagen, pro-
brates was probably of neural crest origin, duced in the ectoderm, can migrate down
i.e., the homologous bones in present day into the underlying mesoderm and there
vertebrates are derived from the cells of the contribute to the extracellular matrix pro-
neural crest. Romer (1972) has divided the duced by the scleroblasts—a present day
skeleton into the predominantly cartilagi- visualization of delamination. The exo- and
nous "visceral skeleton" of neural crest ori- endoskeletons and the various scleroblast
gin and the predominately post-cranial types of the neural crest are thus linked by
"somatic skeleton" of mesodermal origin these common developmental processes.
and has argued that the visceral skeleton They may also be linked, as noted earlier,
antedated the somatic skeleton in verte- with inductive interactions with the under-
brate evolution. The neural crest must then lying nervous tissue (Schowing, I968a,b,c).
have appeared very early in vertebrate
evolution and its cells possessed the proper- Ectodermal and mesodermal scleroblasts
ties of migration, interaction with ec-
toderm, and modulation to different This discussion of the origin of skeletal
scleroblast types equally early. The de- tissues from the neural crest raises the ques-
velopment of epithelial-mesodermal in- tion of the origin of skeletal tissues from the
teractions may have been a later develop- mesoderm and/or the ectoderm, for
ment in vertebrate evolution. Maderson skeletal tissues are traditionally regarded as
(1975) has reviewed the evidence for the mesodermal tissues. As Mathews indicates
homology of developmental processes in (1967, p. 500): "Rigid adherence to re-
 SKELETAL DIFFERENTIATION AND EVOLUTION
quirements for embryological homology
may lead to unnecessary confinement in the
range of phylogenetic considerations."
Romer (1972) provides a delightful anec-
dote on this matter—about the Professor
who delayed publication of his student's re-
search on the ectomesenchymal origin of
the mammalian cranial skeleton for 50
years so as not to "commit treason to the
germ layer theory."
Both ecto- and mesoderm may, and do,
produce the macromolecules characteristic
of connective tissues, viz., collagen and acid
mucopolysaccharides. These two groups of
compounds show co-evolution and, at least
for collagen, conservation of amino acid
structure (Mathews, 1971), although Mat-
sumura (1972) has concluded that amino
acid composition is more tissue-related
than phylogeny-related. Some controversy
exists as to whether vertebrate ectodermal
collagen is different from mesodermal col-
lagen (Moss, 1963, 1970; Mossetal., 1964),
or the same (Matsumura, 1972). Various
invertebrates (e.g., squids, tunicates, and
Branchiostoma) form true epidermal carti-
lage, and some produce composite mus-
culoskeletal tissues as in the intimate associ-
ation of muscle and cartilage in the odon-
tophoral cartilage of grazing gastropods
(Person and Philpott, I969a,b; Carriker et
al., 1972).
Thus, not only can ectodermal cells pro-
duce the macromolecules characteristic of
skeletal tissues, they can also export them to
the mesoderm and so co-participate in
skeletogenesis, or they can elaborate them
locally into skeletal tissues. Moss (I968b,c)
has extended this notion of skeletogenesis
from either germ layer to the development
and evolution of the dermal skeleton of the
ancient vertebrates, especially to the ques-
tion of the origin of vertebrate skeletal tis-
sues during the evolution of the vertebrates
from an invertebrate stock.
INVERTEBRATE ORIGINS OF THE VERTEBRATE
SKELETON
There is an essential unity in mineraliza-
tion mechanism throughout the animal
kingdom (and perhaps also in the plant
kingdom) (Travis et al., 1967; Moss,
341
1968a,c) and although we consider
mineralization of a skeleton to be a verte-
brate prerogative, two thirds of the living
species which contain mineralized tissues
are invertebrates. The origin of vertebrate
calcined tissues from invertebrate ancestors
probably did not require the evolution of
fundamental new biochemical mecha-
nisms, but the selection and modification of
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the appropriate ones from the wide range
of highly developed calcification
mechanisms possessed by the invertebrates
(Mathews, 1967; Person and Philpott,
I969a,b; Philpott and Person, 1970). Cal-
cification in both vertebrates and inverte-
brates occurs in a fibrous matrix. The fibers
are often collagen in invertebrates, always
collagen in vertebrates. The mineral is
CaPO4 in vertebrates, but more often
CaCCh in invertebrates. The invertebrates
are able to mineralize tissues of either ec-
todermal (mollusc shells) or mesodermal
(echinoderm skeletons) origin but have not
evolved mechanisms of interactions be-
tween the two germ layers.
Moss (1968c) has enumerated four pro-
cesses required to produce vertebrate
skeletal tissues which are not possessed by
invertebrates and which therefore must
have evolved very early in the evolution of
vertebrate stock. His four processes may be
simplified to three: (i) formation of a com-
bined (ecto-mesodermal) calcified tissue
with active participation by the ectoderm;
(ii) induction between the mesoderm or ec-
tomesenchyme and the ectoderm; (iii) de-
lamination. The acquisition of the hard
skeleton by the first vertebrates potentially
could have occurred quite rapidly for the
scleroblasts respond very quickly to func-
tional demand.
EPIGENETIC REGULATION OF SCLEROBLAST AC-
TIVITY
Recent studies on the formation of sec-
ondary cartilage on the membrane bones of
the avian skull will serve to illustrate one
environmental factor which can drastically
alter scleroblast behavior and tissue dif-
ferentiation. The environmental factor in-
volved is the presence or absence, and
amount, of movement acting at articular
342 BRIAN K. HALL

surfaces between membrane bones and
other skeletal elements. In the normal
course of embryonic development, carti-
lage arises on membrane bones at their ar-
ticular surfaces late in the 10th or early in
the 11th day of incubation. This coincides
with the time of increased overall move-
ment by the embryo (Hamburger et al.,
1965). If the embryos are paralyzed, or if
possess a narrow range of tissue types the
potential to form other tissue types has not
been lost but is dormant. The tissues
formed in the repair of fractured in-
tramembraneous or endochondral bones
(Hall and Jacobson, 1975; Pritchard and
Ruzicka, 1950; Murray, 1954) are not the
same as the normal skeletal tissues of that
bone or species. Intermediate tissues not

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the membrane bones are cultured in a characteristic of the species arise in bone
stationary culture, cartilage fails to form disease, such as osteosarcoma or chondro-
(Hall, 1967, 1968, 1972; Murray and sarcoma. Bone forms in "cartilaginous"
Smiles, 1965). If mechanical stress is fishes. The primitive scleroblast (both onto-
applied to these bones in culture, cartilage genetically and phylogenetically) is an ex-
can be induced, the amount of cartilage tremely adaptive cell with perhaps more
varying in proportion to the duration of the hidden potential than most other cell types.
stimulus (Hall, 1968). The interesting A study of the skeletal tissues of the an-
finding is that the cartilage which forms in cient vertebrates which neglects such mod-
the 1 lth day, and the bone which was form- ifying epigenetic factors is at the very least
ing earlier and which continues to form incomplete, and at worst inaccurate.
after 11 days, arise from a common pool of
mesenchymal cells. Where the cells are pro-
tected from the mechanical stimulus os-
teogenesis continues. Where the cells are Cellular and acellular bone
mechanically stressed they become chon-
drogenic, and they can, if the stress is in- The views on the relationships between
termittently applied and removed, alter- cellular and acellular Agnathan bone dis-
nate between the two scleroblast (Chondro- cussed earlier take on a new dimension
or Osteoblasts) types (Murray and Smiles, when viewed in the light of the develop-
1965). Alterations in mechanical stresses ment of the acellular bone of modern
were extremely important in the evolution higher teleosts. Such acellular bone is
of the jaws and in the evolution of the entire found in both freshwater and in saltwater
vertebrate head and, as these recent exper- species and so appears not to be correlated
iments indicate, mechanical stress is a po- with retention of calcium or of phosphor-
tent modulator of scleroblast behavior. ous (Moss, 1961a, 1965). The functional
Moss (1969) has shown that, within the significance of acellular bone remains un-
reptilian dermis, a wide range of skeletal known. The acellular bone of the teleosts
tissues may be produced from similar pre- develops in one of three ways from one of
sumptive cells. For example, all stages from three types of cellular tissues: periosteal os-
tendon, through calcified tendon to bone, teogenesis from the cells (osteoprogenitors)
are found, and he has argued (1964a) that of the periosteum; tendonous osteogenesis
most primitive vertebrates also had the po- from the cells within the tendons; or os-
tential to produce the whole range of teogenesis by metaplasia of secondary car-
skeletal tissues during their evolution- tilage cells (Moss, 1964a). Thus, Moss
selection pressure, and modulation dictat- (19646, p. 348) writes: "It is apparent that
i ng which type of tissue would actually form no neccessary relationship exists between
at a given site in a given species at a given the type of bone first formed, and the type
time. The restriction in the range of skeletal which is eventually characteristic of the
tissues as one ascends the vertebrate species." The osteocytes so-formed either
phylogenetic tree indicates that particular are not enclosed in an osseous matrix or, if
skeletal tissues have become preferentially enclosed, are replaced by an osseous
selected for during the course of evolution. matrix, resulting in an acellular bony
However, even in those vertebrates which matrix in the adult. Similar transforma-
 SKELETAL DIFFERENTIATION AND EVOLUTION 343

tions of cytoplasm into supporting tissue
matrix are seen in some invertebrate carti-
lages and in lignification in plants (Person
and Philpott, 1963).
It is difficult to imagine how the acellular
bone of individual Ordovician Agnatha
could have developed without going
through a cellular phase. Thus, when we
evaluate the evidence for the possible
ossification within long bones likewise in-
volved progressive adaptation of pre-
existing mechanisms of chondro- and os-
teogenesis, rather than the development of
new mechanisms (discussed by Moss,
1964a). The limitations placed upon the
evolution of the skeletal tissues must then
be assessed on the basis of the differentia-
tive potential of the constitutent cells of the

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evolution of acellular bone (aspidin) from skeleton.
cellular dermal bone we should really be To summarize the answer to the question
considering the origin of cellular bone of how the various skeletal tissue types form
from cellular bony or other connective tis- at specific sites, it appears that the spec-
sues. The important evolutionary question ificity comes from local environmental fac-
is then really a developmental question: tors such as contact with specific epithelia,
Why do the osteocytes not persist? (0rvig, local mechanical stresses, and not always
1957,1965; Calder and Hall, unpublished). from intrinsic predetermination of the po-
tentially skeletogenic cells. (The cells are
Sesamoids and secondary centers of ossification biased toward skeletogenesis, but epigenetic
factors determine which particular sclero-
Some structures which are almost uni- blast type will develop at a given site.) These
versally present in the long bones of recent cells are then potentially and actually inter-
mammals, such as secondary centers of os- convertible and the consequences of such
sification within the epiphyses, or sesamoid interactions and modulations must be kept
bones at joints, appear to have arisen with in mind when considering the evolution of
the mammals in the Jurassic (Haines, skeletal tissues and especially when basing
1969). Do these not indicate the progressive phylogeny on tissue and cell structure.
evolution of skeletal tissue structure? Ap-
parently not, if we look at the development
of such structures. Sesamoid bones are SIZE AND SHAPE OF THE SKELETON
known to be intratendinous ossifications
(Barnet and Lewis, 1958) or intra- We may now turn to the question: What
ligamentous ossifications (Burton, 1973) determines the size and shape of the skele-
and it is known that the ornithischian di- ton?
nosaurs possessed ossified tendons Morphogenesis of the skeleton
(Haines, 1969) and that reptiles tend to
show tendinous rather than periosteal os- Once the cells of the skeleton begin to
sification (Moss, 1969; Enlow, 1969). The differentiate a three-dimensional structure
ligaments and tendons arise from connec- develops. At first the cells within the con-
tive tissue along lines of stress (Schaeffer densed mesoderm are randomly arranged.
and Rosen, 1961) and local tensions appar- As the cells begin to synthesize extracellular
ently induce them to ossify (Haines and matrix and to differentiate, they elongate
Mohuidiin, 1968). Thus, the formation of perpendicular to the Jong axis of the con-
the sesamoids need not have involved the densation, and so begin to initiate a pattern
development of radically new processes of and direction to the growth and shape of
ossification, and they are not bones radi- the ruminent. From then on in the de-
cally different in construction from other velopment of the skeletal rudiment factors
bones. Their evolution probably involved
the adaptation of pre-existing processes in other than the direct genetic constitution of
response to new environmental conditions the tissues begin to come into play.
(the extra stresses imposed on the joints by A study of the determination of the mor-
the adoption of terrestrial life). phology of particular bones within the
skeleton sheds considerable light on the rel-
The formation of secondary centers of ative contributions of genetic and
344 BRIAN K. HALL
"epigenetic" factors. Thompson (1917), in
a now classic study, considered the mainte-
nance of form in the animal and plant
kingdoms as an adaptation to the environ-
ment but avoided the question of the rela-
tionship between ontogenetic adaptation
and the inheritance of form and pattern.
Murray (1936) summarized the early litera-
ture on the form of bones, most of which
was based on the trajectory theory of Wolff,
viz., that the form of the bone is, in large
measure, molded by the external forces act-
ing upon it. Enlow (1968) has provided a
recent evaluation of Wolffs law. It turns
out that inherent genetic control is more
important than was previously thought to
be the case.
The attainment of the fundamental form
(i.e., the initial three-dimensional mor-
phology, accompanied by considerable
linear growth) of a skeletal element is inde-
pendent of functional demands, and is
under genetic control (Howell, 1917; Felts,
1961; Chalmers and Ray, 1962; Mawdsley
and Ainsworth Harrison, 1963). For exam-
ple, if the mesodermal primordium of a
bone, or the early bony anlage after initia-
tion of osteogenesis or chondrogenesis, is
grafted to the chorio-allantoic membrane,
or transplanted subcutaneously or in-
tramuscularly, or cultivated in vitro (even
in the presence of unusually strong
mechanical stresses) this fundamental
form—three-dimensional configuration,
presence and position of condyles,
tuberousities and grooves—develops nor-
mally (Murray, 1926, 1928; Monson and
Felts, 1961; Hall, 1967, 1968; Ede, 1971;
Yasuda, 1973). There is expression of the
inherent rates of cell division, cell hyper-
trophy, and amounts of intercellular
matrix produced per cell.
Once this fundamental form is estab-
lished, the development of "minor" ar-
chitectural features of the bone (ridges for
attachment of muscles, ligaments etc.) de-
pends upon functional demand and can be
modified by the environment (Murray,
1936; Chalmers, 1965; Drachman and
Sokoloff, 1966). The appearance of these
"minor" architectural features establishes
the final form of the bone, and the con-
tinued action of mechanical factors is nec-
cessary to maintain that form.
The evolutionary consequences of these
studies with growing bones would seem to
be that to change the morphogenetic pro-
cesses which are responsible for the basic
three-dimensional form of the bone, would
require considerable, integrated alteration
in the genome and so would be a relatively
slow phylogenetic process. Changes in the
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minor elements of the bone's form could
occur quite rapidly as ontogenetic mod-
ifications within one lifetime.
Functional units and epigenetic factors
Gruneberg (1963) and Moss (1968d)
have reviewed the evidence which indicates
that the final position, shape, size, and
growth of particular skeletal elements are
in large measure secondary responses to
the functional unit (lower jaw, skull, upper
arm, etc.) of which the skeletal element is a
part. That is, organs and tissues adjacent to
the skeleton modify its pattern of growth
and determine (through tissue-tissue in-
teractions) whether the cells destined to
form the tissues of the skeleton will dif-
ferentiate at all, and further, what type of
tissue they will form. Moss (1968rf) goes so
far as to say that: "It is incorrect to speak of
the evolution of the skeleton as such [for] it
is the functional matrix which evolves, the
bone only responds." I would agree, pro-
vided that only the minor features of the
skeleton are included. Some examples
from recent experimental studies are pro-
vided below.
If the vitreous humor is drained from the
eye(s) of the embryonic chick late in the 4th
day of incubation, microphthalmia is in-
duced and the growth of the eye slowed
down. If the embryo is examined at 18 days
of incubation, the eye is found to be smaller
than normal. The size, shape, and position
of the orbital bones adjacent to the eye are
also found to deviate from normal. Furth-
ermore, bones further removed from the
orbit, such as the frontal, are also found to
be abnormal (Coulombre and Crelin,
1958). Thus, the growth of the eye exerts a
considerable influence on the mor-
phogenesis, growth, and pattern formation
of the adjacent and subjacent skeletal ele-
 SKELETAL DIFFERENTIATION AND EVOLUTION 345

merits which form parts of the same func-
tional unit.
The muscles of the head also play a role
in controlling the growth of this functional
unit. Bilateral masseterectomy in the new-
born rat reduces the size of the facial and
cranial bones and it does so asymmetrically,
one dimension of the bones being more
affected than the others (Moore, 1967).
viously unconnected skeletal elements as
described above often leads to the de-
velopment of a new articulation, or to the
modification of existing sutures, joints, or
articulations to accommodate the evolution
of the complex musculo-skeletal-
connective tissue functional unit. The basic
(primitive?) suture between two adjacent
bones is what has been termed the flat suture

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However, the basic architectural features of (Moss, 1957) and consists of two bone sur-
the bones are unaffected by surgical ma- faces opposed to one another without in-
nipulations to the adjacent soft tissues terdigitation. Such sutures may be mod-
(Pratt, 1943). ified in response to the functional demand
The types of tissues produced at a given made on them and develop interdigita-
site within the skeleton, the association be- tions, overlapping surfaces, etc. Such is the
tween adjacent skeletal units, and the plas- case in the skull of the woodpecker and
ticity of skeletal tissues for developmental provides an answer to the enigmatic ques-
modulations and responses to environmen- tion: "Why don't woodpeckers get
tal stimulii during evolution are amply illus- headaches?" It turns out that the suture
trated by the following studies. Bock between the frontal and the nasal bones is
(196CM) and Bock and Morioka (1968) an overlapping one, enabling one bone to
have carried out a series of studies, illustrat- ride over the other and absorb some of the
ing the repeated evolution of elements of stresses which would otherwise be directed
the avian skull. For example, the palatine onto the articulation. This implies changes
process of the premaxilla may be either in the soft tissues associated with the articu-
fused or unfused and has been lost and lations related to functional demand, and
reappeared many times during avian although evidence is difficult to obtain from
evolution (presumably in response to newly the fossil record, numerous experimental
appearing stresses and pressures). The examples are available: for example, the
median process of the mandible has presence or absence of, and the degree of
likewise appeared where stress on the fibrous development in, the intra-articular
mandible and strength of the quadrate dic- discs of the temporo-mandibular joints of
tated (Bock, 19606), a situation which can Marsupials and Monotremes (Sprinz,
be induced experimentally by paralysis of 1965), or the transformation of fibrous ar-
avian embryos (Murray and Drachman, ticular tissues to fibrocartilage to allow the
1969). Simonetta (1960) and Bock (1964) human temporo-mandibular joint to adapt
have reviewed the question of the evolution morphologically to mechanical stresses
of the kinetic avian skull and the factors (Moffett et al., 1964).
which modify its skeletal elements of the
skull. What are the implications of these studies
for establishment of phylogenetic trends in
Single character analyses such as those of skeletal tissues?
Bock and of Beecher (1950, 1951) and
Cracraft (1968) serve to show the wide vari- Phytogeny of skeletal tissues
ation in morphology which may exist be-
tween closely related species and highlight These studies illustrate the difficulties in-
that there are many ways by which skeletal volved in establishing a phylogeny of
elements may respond to environmental skeletal tissues based on the genetic selec-
conditions. The final form of the character tion of progressively more well-adapted tis-
depends upon (at least): (i) selection acting sue types. A partial list of the epigenetic
directly on the character; (ii) influences factors which can influence skeletal his-
from neighboring structures; (iii) chance togenesis and final structure and which
factors (Cracraft, 1968). presumably did so early in vertebrate
The establishment of contact of two pre- evolution would include: rate of growth of
346 BRIAN K. HALL

the bone, rate of growth of the rest of the the vertebrate lineage, make such attempts
functional unit, degree of remodeling of difficult, and indeed, there may be no basis
the bone, muscle attachments, degree of in fact for such evolutionary trends. The
vascularization, mechanical factors, size of earliest vertebrates possessed highly
the animal, habitat (terrestrail, aquatic, ae- specialized skeletal tissues and left evi-
rial), length of the development period, age dence, in the form of tissues intermediate
at maturity, seasonal feeding cycles, and between those recognized as discrete end-
hormonal milieu. points (bone, cartilage, dentine) that their
When we say that no major structural cells were highly adaptive even at the outset

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advances were associated with the evolution of vertebrate evolution.
of the skeleton and that a phylogeny of The readiness with which skeletal tissues
bone or of cartilage, involving progressive respond to epigenetic factors provides con-
advancement with time cannot be estab- siderable insight into the mechanisms
lished, we do not wish to imply that no whereby the skeleton and its associated soft
changes occurred with time. Moss (1964a) tissues may have evolved. The skeletal tis-
has listed four trends in the evolution of the sues are supremely pre-adapted to exploit
skeleton: decrease in the range of skeletal new environmental pressures. Con-
types; decrease in the amount of bone per vergence and divergence ought to be ex-
animal; decrease in the number of bones pected and according to Jollie (1968) oc-
per animal; and a more restricted location curred in the initial, separation of the
of bone (e.g., decrease in dermal armor). gnathostomes from the agantha. The
Note that none of these involve increased modulation of the scleroblasts to cartilage
specializations of the skeletal tissues; if any- facilitated rapid embryonic growth. Mod-
thing they reflect dimunition of skeletoge- ulation to bone facilitated storage of essen-
nesis with time. Moss also lists three factors tial ions, bearing of increased weight, and
associated with evolution above the fish the transition to land. These are some of
level which he considers as possibly respon- the evolutionary consequences of skeletal
sible for the above changes: the skeleton differentiation.
becomes weight bearing, homeostatic for
calcium and phosphorous, and a source of NOTE ADDED IN PROOF
hematopoeitic cells.
Recently, le Lievre (1974) has shown that
both bone and cartilage of the avian visceral
CONCLUDING COMMENTS skeleton are derived from ectomesenchyme
of the neural crest.
The skeletal tissues of both fossil and re-
cent vertebrates form a coherent, interre-
lated class, according to a number of
criteria: embryological development, pro- REFERENCES
duction of extracellular matrices consisting
of a fibrous protein and a carbohydrate Alexander, R. McN. 1975. Evolution of integrated
component, ability to calcify the matrix, the design. Amer. Zool. 15:419-425.
potential to modulate to other cell types, Applegate, S. P. 1967. A survey of shark hard parts.
Pages 37-67 in P. W. Gilbert, R. F. Mathewson, and
high degree of responsiveness to modifica- D. P. Rail, eds., Sharks, skates and rays. Johns Hop-
tion by environmental factors. There is, kins Press, Baltimore.
therefore, justification in speaking of Barnett, C. H., and O. J. Lewis. 1958. The evolution of
skeletal tissues as closely related to one some traction epiphyses in birds and mammals. J.
Anat. 92:593-601.
another and this has led to attempts to es- Beecher, W. J. 1950. American Orioles. Wilson Bull.
tablish phylogenies of skeletal tissues. The 62:51-86.
data presented in this paper indicate that Beecher, W. J. 1951. Adaptation to food-getting in the
the very high degree of plasticity shown by American blackbirds. Auk 68:411-440.
these cells, their readiness to modulate in Beer, G. de. 1958. Embryos and ancestors. 3rd ed.
response to epigenetic factors, and their Berrill, Oxford Univ. Press, London.
N.J. 1955. The origin of the vertebrates. Ox-
early appearance as specialized tissues in ford Univ. Press, London.
 SKELETAL DIFFERENTIATION AND EVOLUTION 347

Biltz, R. M., and E. D. Pellagrino. 1969. The chemical 54:803-822.

anatomy of bone. 1. A comparative study of bone Enlow, D. H. 1969. The bone of reptiles. Pages 45-80
composition in sixteen vertebrates. J. Bone Joint in C. Cans, ed., Biology of the Reptilia. Vol. 1.
Surg. 51A:456-466 Academic Press, New York.
Bock, W. J. 1960a. The palatine process of the pre- Enlow, D. H., and S. O. Brown. 1956. A comparative
maxilla in the Passeres. A study of the variation, histological study of fossil and recent bone tissues.
function, evolution and taxonomic value of a single Part I. Texas J. Sci. 8:405-443.
character throughout an avian order. Bull. Mus. Enlow, D. H., and S. O. Brown. 1957. A comparative
Comp. Zool. 122:361-488. histological study of fossil and recent bone tissues.
Bock, W. J. 19606. Secondary articulation of the avian Part II. Texas J. Sci. 9:186-214.

Downloaded from https://academic.oup.com/icb/article/15/2/329/2126723 by guest on 24 September 2020

mandible. Auk 77:19-55. Enlow, D. H. and S. O. Brown. 1958. A comparative
Bock, W. J. 1964. Kinetics of the avian skull. J. Mor- histological study of fossil and recent bone tissues.
phol. 114:1-42. Part III. Texas J. Sci. 10:187-230.
Bock, W. J., and H Morioka. 1968. The ecthemoid- Felts, W. J. L. 1961. In vivo implantation as a technique
mandibular articulation in some Meliphagidae in skeletal biology. Int. Rev. Cytol. 12:243-302.
(Aves). Amer. Zool. 8:808. (Abstr.) Felts, W. J. L., and F. A. Spurrell. 1966. Some struc-
Bock, W. J., and G. von Wahlert. 1965. Adaptation tural and developmental characteristics of cetacean
and the form-function complex. Evolution 19:269- (Odontocete) radii. A study of adaptive os-
299. teogenesis. Amer. J. Anat. 118:103-134.
Brul, E. L. du. 1964. Evolution of the temporoman- Friant, M. 1959. Sur l'ossification enchondrale du car-
dibular joint. Pages 3-27 in B. G. Sarnat, ed., The tilage de Meckel chez les Rongeurs. Bull. GR. Int.
temporomandibular joint. C. C. Thomas, Spring- Res. Sci. Stomatol. 4:1-11.
field, 111. Friant, M. 1964. Sur l'ossification du cartilage de Mec-
Burton, P. J. K. 1973. Structure of the depressor man- kel d'une chauvre-souris, le grand Murin (Chirop-
dibulae muscle in the Kokako Callaeas cinerea. Ibis tera), Myotis myotis (Borkh). Acta Anat. 57:66-71.
115:138-139. Friant, M. 1966. Vue d'ensemble sur revolution du
Carriker, M. R., P. Person, R. Libbin, and D. van "cartilage de Meckel" de quelques groupes de
Zandt. 1972. Regeneration of the proboscis of Mammiferes. Acta Zool. 47:67-80.
muricid gastropods after amputation, with em- Friant, M. 1968. L'evolution de cartilage de Meckel du
phasis on the radula and cartilages. Biol. Bull. pore (Sus scrofa dom., Gray). Ann. Fac. Med. Vet.
143:317-331. Pisa 21:1-17.
Chalmers, J. 1965. A study of some of the factors Friant, M. 1969. L'evolution du cartilage de Meckel du
controlling growth of transplanted skeletal tissue. cheval (Equus caballus L.). Ann Fac. Med. Vet. Pisa
Pages 177-184 in L. J. Richelle and M. J. Dal- 22:253-275.
lemagne, eds., Proc. 2nd Eur. Symp. Calcified Tis- Frommer.J., and M. R. Margolies. 1971. Contribution
sues. Coll. des Colloq. de l'Univ. de Liege. of Meckel's cartilage to ossification of the mandible
Chalmers, J., and R. D. Ray. 1962. Transplantation in mice. J. Dent. Res. 50:1260-1267.
immunity in bone homografting. J. Bonejoint Surg. Goedbloed, J. F. 1964. The early development of the
44B:149-164. middle ear and the mouth cavity. A study of the
Coulombre, A. J., and E. S. Crelin. 1958. The role of interaction of processes in the eqithelium and the
the developing eye in the morphogenesis of the mesenchyme. Arch. Biol. 75:207-244.
avian skull. Amer. J. Phys. Anthropol. 16:25-37. Griineberg, H. 1963. The pathology of development.
Cracraft, J. 1968. The lacrimal-ectethmoid bone com- A study of inherited skeletal disorders in animals.
plex in birds: a single character analysis. Amer. Mid- Blackwell Scientific Publications, Oxford.
land Natur. 80:316-359. Haines, R. W. 1969. Epiphyses and sesamoids. Pages
Crelin, E. S., and W. E. Koch. 1967. An autoradiogra- 81-116 in C. Cans, ed., Biology of the Reptilia.
phic study of chondrocyte transformation into Academic Press, New York.
chondroclasts and osteocytes during bone forma- Haines, R. W., and A. Mohuiddin. 1968. Metaplastic
tion in vitro. Anat. Rec. 158:473-484. bone. J. Anat. 103:527-538.
Denison, R. H. 1963. The early history of the verte- Hall, B. K. 1967. The formation of adventitious carti-
brate calcified skeleton. Clin. Orthop. Related Res. lage by membrane bones under the influence of
31:141-152. mechanical stimulation applied in vitro. Life Sci.
Drachman, D. B., and L. Sokoloff. 1966. The role of 6:663-667.
movement in embryonic joint development. De- Hall, B. K. 1968. In vitro studies on the mechanical
velop. Biol. 4:401-420. evocation of adventitious cartilage in the chick. J.
Ede, D. A. 1971. Control of form and pattern in the Exp. Zool. 168:283-306.
vertebrate limb. Symp. Soc. Exp. Biol. 25:235-254. Hall, B. K. 1970. Cellular differentiation in skeletal
Enlow, D. H. 1966. An evaluation of the use of bone tissues. Biol. Rev. Camb. Philos. Soc. 45:455-484.
histology in forensic medicine and anthropology. Hall, B. K. 1971. Histogenesis and morphogenesis of
Pages 93-112 in F. G. Evans, ed., Studies on the bone. Clin. Orthop. Related Res. 74:249-268.
anatomy and function of bone and joints. Hall, B. K. 1972. Immobilization and cartilage trans-
Springer-Verlag, Heidelberg. formation into bone in the embryonic chick. Anat.
Enlow, D. H. 1968. Wolffs law and the factor of ar- Rec. 174:391-403.
chitectonic circumstance. Amer. J. Orthodont. Hall, B. K., and H. N. Jacobson. 1975. The repair of
348 BRIAN K. HALL
fractured membrane bones in the newly hatched
chick. Anat. Rec. 181:55-70.
Halstead, L. B. 1969a. Calcified tissues in the earliest
vertebrates. Calcified Tissue Res. 3:107-124.
Halstead, L. B. 196%. The pattern of vertebrate
evolution. Oliver and Boyd, Edinburgh.
Hamburger, V., M. Balaban, R. Oppenheim, and E.
Wenger. 1965. Periodic motility of normal and spi-
nal chick embryos between 8 and 17 days of incuba-
tion. J. Exp. Zool. 159:1-14.
Hammond, W. S.,andC. L. Yntema. 1964. Depletions
of pharyngeal arch cartilages following extirpation
of cranial neural crest in chick embryos. Acta Anat.
56:21-34.
Hancox, N. M. 1972. Biology of bone. Cambridge
Univ. Press, Cambridge.
Hay, E. D. 1964. Secretion of a connective tissue pro-
tein by developing epidermis, Pages 97-116 in W.
Montagna and W. Lobitz, Jr., eds., The epidermis.
Academic Press, New York.
Ho, T. Y. 1967. The amino acids of bone and dentine
collagens in Pleistocene mammals. Biochim.
Biophys. Acta 133:568-573.
Holtfreter, J. 1968. Mesenchyme and epithelia in in-
ductive and morphogenetic processes. Pages l-30m
R. Fleischmajer, ed., Epithelia-mesenchymal in-
teractions. Williams and Wilkins, Baltimore.
Holtrop, M. E. 1966. The origin of bone cells in en-
dochondral ossification. Calcined Tissues, Proc.
Eur. Symp. 3:32-36.
Holmgren, N. 1940. Studies on the head infishes.Acta
Zool. 21:51-267.
Holmgren, N. 1942. Studies on the head in fishes. Part
III. The phylogeny of elasmobranch fishes. Acta
Zool. 23:129-261.
Horstadius, S. 1950. The neural crest. Oxford Univ.
Press, London.
Howell, J. A. 1917. An experimental study of the ef-
fects of stress and strain on bone development.
Anat. Rec. 13:233-252.
Jacobson, W., and H. B. Fell. 1941. The developmen-
tal mechanics and potencies of the undifferentiated
mesenchyme of the mandible. Quart. J. Microscop.
Sci. 82:563-586.
Jarvik, E. 1959. Dermal fin-rays and Holmgren's prin-
ciple of delamination. Kgl. Svenska Vetenskapsakad
Handl. 6:3-49.
Johnson, M. C. 1966. A radioautographic study of the
migration and fate of cranial neural crest cells in the
chick embryo. Anat. Rec. 156:143-156.
Johnson, M. C., and M. A. Listgaren. 1972. Observa-
tions on the migration, interaction, and early dif-
ferentiation of orofacial tissues. Pages 55-80 in H. C.
Slavkin and L. A. Bavetta, eds., Developmental as-
pects of oral biology, Academic Press, New York.
Jollie, M.C. 1968. Some implications of the acceptance
of a delamination principle. Pages 89-107 in T. 0r-
vig, ed., Current problems of lower vertebrate
phylogeny. Almquist and Wiksell, Stockholm.
Jollie, M.C. 1971. Some developmental aspects of the
head skeleton of the 35-37mm Squalus acanthias
foetus. J. Morphol. 133:17-40.
Kobayashi, S. 1971. Acid mucopolysaccharides in cal-
cified tissues. Int. Rev. Cytol. 30:257-371.
Koch, W. E. 1972. Tissue interaction during in vitro
odontogenesis. Pages 151-164 in H. C. Slavkin and
L. A. Bavetta, eds., Developmental aspects of oral
biology. Academic Press, New York.
Kolliker, A. 1859. On the different types in the micro-
structure of the skeleton of the osseous fishes. Proc.
Roy. Soc. London 9:656-668.
Lash, J. W. 1968. Chondrogenesis: genotypic and
phenotypic expression. J. Cell Physiol. 72 (Suppl.
l):35-46.
le Lievre, C. 1974. Role des cellules mesectodermiques
issues des cretes neurales cephaliques dans la for-
Downloaded from https://academic.oup.com/icb/article/15/2/329/2126723 by guest on 24 September 2020
mation des arcs branchiaux et du squelette visceral.
J. Embryol. Exp. Morphol. 31:453-477.
McLean, F. C, and M. R. Urist. 1968. Bone. Funda-
mentals of the physiology of skeletal tissues. 3rd ed.
Univ. Chicago Press, Chicago.
MacConaill, M. A. 1973. Calcopheritic calcification of
cartilage. J. Anat. 115:23-28.
Maderson, P. F. A. 1975. Embryonic tissue interac-
tions as the basis for morphological change in evolu-
tion. Araer. Zool. 15:315-327.
Mereel, M. 1967. Recherches sur la relation inductrice
entre chondrocytes et perioste dans le tibia era-
bryonnaire du poulet. Arch. Biol. 78:145-166.
Mathews, M. B. 1966. The molecular evolution of
cartilage. Clin. Orthop. Related Res. 48:267-283.
Mathews, M. B. 1967. Macromolecular evolution of
connective tissues. Biol. Rev. Camb. Philos. Soc.
42:499-551.
Mathews, M. B. 1971. Comparative biochemistry of
chondroitin sulphate-proteins of cartilage and
notochord. Biochem. J. 125:37-46.
Matsumura, T. 1972. Relationship between amino
acid composition and differentiation of collagen.
Int. J. Biochem. 3:265-274.
Mawdsley, R.,andG. A. Harrison. 1963. Environmen-
tal factors determining the growth and develop-
ment of whole bone transplants. J. Embryol. Exp.
Morphol. 11:537-547.
Moffett, B. C, Jr., L. C.Johnson, J. B. Macabe, and H.
C. Askew. 1964. Articular remodelling in the adult
human temporomandibular joint. Amer. J. Anat.
115:119-142.
Monson, J. W., and W. J. L. Felts. 1961. Transplanta-
tion studies of factors in skeletal organogenesis. II
The response of the immature mouse humerus to
longitudinal compressive forces. Amer. J. Phys. An-
thropol. 19:63-67.
Moore, W. J. 1967. Muscular function and skull
growth in the laboratory rat (Rattus norvegicus). J.
Zool. (London) 152:287-296.
Moss, M. L. 1957. Experimental alteration of sutural
area morphology. Anat. Rec. 127:569-590.
Moss, M. L. 1961a. Studies on the acellular bone of
teleost fish. 1. Morphological and systematic varia-
tion. Acta Anat. 46:343-362.
Moss, M. L. 19616. The initial phylogenetic appear-
ance of bone: an experimental hypothesis. Trans. N.
Y. Acad. Sci. 23:495-500.
Moss, M. L. 1963. The biology of acellular teleost
bone. Ann. N. Y. Acad. Sci. 109:337-350.
Moss, M. L. 1964a. The phylogeny of mineralised
tissues. Int. Rev. Gen. Exp. Zool. 1:297-331.
Moss, M. L. 19646. Development of cellular dentin and
lepidosteal tubules in the bowfin, Amia calva. Acta
 SKELETAL DIFFERENTIATION AND EVOLUTION
Anat. 58:333-354.
Moss, M. L. 1965. Studies of the acellular bone of
teleost fish. V. Histology and mineral homeostasisof
fresh-water species. Acta Anat. 60:262-276.
Moss, M. L. 1968a. Bone, dentin and enamel and the
evolution of vertebrates. Pages 37-65 in P. Person,
ed., The biology of the mouth. Amer. Ass. Advan.
Sci., Washington, D.C.
Moss, M. L. 1968A. Comparative anatomy of verte-
brate dermal bone and teeth. I. The epidermal co-
participation hypothesis. Acta Anat. 71:178-208.
Moss, M. L. 1968c. The origin of vertebrate calcified
tissues. Pages 359-371 in T. 0rvig, ed., Current
problems of lower vertebrate phylogeny. Almquist
and Wiksell, Stockholm.
Moss, M. L. 1968d. Functional cranial analysis of
mammalian mandibular ramal morphology. Acta
Anat. 71:423-447.
Moss, M. L. 1969. Comparative histology of dermal
sclerifications in reptiles. Acta Anat. 73:510-533.
Moss, M. L. 1970. Enamel and bone in shark teeth:
with a note on fibrous enamel in fishes. Acta Anat.
77:161-187.
Moss, M. L. 1972a. The vertebrate dermis and the
integumental skeleton. Amer. Zool. 12:27-34.
Moss, M. L. 19726. The regulation of skeletal growth.
Pages 127-141 in R.J. Goss.ed., Regulation of organ
and tissue growth. Academic Press, New York.
Moss, M. L., S.Jones, and K. A. Piez. 1964. Calcified
ectodermal collagens of shark tooth enamel and
teleost scale. Science 145:940-942.
Murray, P. D. F. 1926. An experimental study of the
development of the limbs of the chick. Proc. Lin-
nean Soc. N.S.W. 51:187-263.
Murray, P. D. F. 1928. Chorio-allantoic grafts of frag-
ments of the two-day chick, with special reference to
the development of the limbs, intestine and skin.
Aust. J. Exp. Biol. Med. Sci. 5:237-256.
Murray, P. D. F. 1936. Bones. Cambridge Univ. Press,
Cambridge.
Murray, P. D. F. 1954. The fusion of parallel long
bones and the formation of secondary cartilage.
Aust. J. Zool. 2:364-380.
Murray, P. D. F.,and D. B. Drachman. 1969. The role
of movement in the development of joints and re-
lated structures: the head and neck in the chick
embryo. J. Embryol. Exp. Morphol. 22:349-371.
Murray, P. D. F., and M. Smiles. 1965. Factors in the
evocation of adventitious (secondary) cartilage in
the chick embryo. Aust. J. Zool. 13:351-381.
Noble, H. W. 1973. Comparative functional anatomy
of temporomandibular joint. Oral Sci. Rev. 2:3-28.
0rvig, T. 1951. Histological studies of placoderms and
fossil elasmobranchs. I. The endoskeleton with re-
marks on the hard tissues of lower vertebrates in
general. Arkiv. Zool. 2:321-456.
0rvig, T. 1957. Palaeohistological notes. I. On the
structure of the bone tissue in the scales of certain
Palaeonisciformes. Ark. Zool. 10:481-490.
0rvig, T. 1965. Palaeohistological notes. 2. Certain
comments on the phyletic significance of acellular
bone tissue in early vertebrates. Ark. Zool. 16:551-
556.
0rvig, T. 1967. Phylogeny of tooth tissues: evolution
of some calcified tissues in early vertebrates. Pages
349
45-110 m A. E. W. Miles, ed., Structural and chemi-
cal organization of teeth. Academic Press, New
York.
0rvig, T. 1968. The dermal skeleton: general consid-
erations. Pages 373-397 in T. 0rvig, ed., Current
problems of lower vertebrate phylogeny. Almquist
and Wiksell, Stockholm.
Person, P., and D. E. Philpott. 1963. Invertebrate car-
tilages. Ann. N. Y. Acad. Sci. 109:113-126.
Person, P., and D. E. Philpott. 1969a. The nature and
significance of invertebrate cartilages. Biol. Rev.
Downloaded from https://academic.oup.com/icb/article/15/2/329/2126723 by guest on 24 September 2020
Camb. Philos. Soc. 44:1-16.
Person, P., and D. E. Philpott. 19696. The biology of
cartilage. I. Invertebrate cartilages: Limulus gill car-
tilage. J. Morphol. 128:67-94.
Peyer, B. 1968. Comparative odontology. Univ.
Chicago Press, Chicago.
Philpott, D. E., and P. Person. 1970. The biology of
cartilage. II. Invertebrate cartilages: squid head car-
tilage. J. Morphol. 131:417-430.
Pratt, L. W. 1943. Experimental masseterectomy in
the laboratory rat. J. Mammalogy 24:204-211.
Pritchard, J. J., and A. J. Ruzicka. 1950. Comparison
of fracture repair in the frog, lizard and rat. J. Anat.
84:236-261.
Ricqles, A. de. 1968. Recherches paleohistologiques
sur les os longs des tetrapodes. I. Origine du tissu
osseux plexiforme des dinosauriens sauropodes.
Ann. Paleontol. 54:133-145.
Ricqles, A. de. 1969. Recherches paleohistologiques
sur les os longs des tetrapodes. II. Quelques obser-
vations sur la structure des os longs des teriodontes.
Ann. Paleontol. 55:3-52.
Ricqles, A. de. 1972. Recherches paleohistologiques
sur les os longs des tetrapods. III. Titanosuchiens,
dinocephales et dicynodontes. Ann. Paleontol.
58:17-60.
Romer, A. S. 1942. Cartilage an embryonic adapta-
tion. Amer. Nautr. 76:394-404.
Romer, A. S. 1963. The ancient history of bone. Ann.
N. Y. Acad. Sci. 109:168-176.
Romer, A. S. 1964. Bone in early vertebrates. Pages
13-37 in H. M. Frost, ed., Bone biodynamics. Little,
Brown and Co., Boston.
Romer, A. S. 1972. The vertebrate as a dual animal-
somatic and visceral. Evol. Biol. 6:121-156.
Schaeffer, B. 1961. Differential ossification in the
fishes. Trans. N. Y. Acad. Sci. 23:501-505.
Schaeffer, B., and D. E. Rosen. 1961. Major adaptive
levels in the evolution of the actinopterygian feed-
ing mechanism. Amer. Zool. 1:187-204.
Schowing, J. 1968a. Influence inductrice de I'en-
cephale embryonnaire sur le developpement du
crane chez le poulet. I. Influence de l'excision des
territoires nerveux anterieurs sur le developpement
cranien.J. Embryol. Exp. Morphol. 19:9-22.
Schowing, J. 19686. Influence inductrice de 1'en-
cephale embryonnaire sur le developpement du
crane chez le poulet. II. Influence de l'excision de la
chorde et des territories encephaliques moyen et
posterieur sur le developpement cranien. J. Em-
bryol. Exp. Morphol. 19:23-32.
Schowing, J. 1968c. Influence inductrice de l'en-
cephale embryonnaire sur le developpement du
crane chez le poulet. III. Mise en evidence du role
 Downloaded from https://academic.oup.com/icb/article/15/2/329/2126723 by guest on 24 September 2020
350 BRIAN K. HALL

inducteur de l'encephale dans l'osteogenese du Urist, M. R. 1962. The bone-body fluid continuum:
crane embryonnaire du poulet. J. Embryol. Exp. calcium and phosphorous in the skeleton and blood
Morphol. 19:83-94. of extinct and living vertebrates. Perspect. Biol.
Sicher, H. 1966. Orban's oral histology and embryol- Med. 6:75-115.
ogy. 6th ed. C. V. Mosby Co., St. Louis. Urist, M. R. 1963. The regulation of calcium and other
Simonetta, A. M. 1960. On the mechanical implica- ions in the serums of hagfish and lampreys. Ann. N.
tions of the avian skull and their bearing on the Y. Acad. Sci. 109:294-311.
evolution and classification of birds. Quart. Rev. Urist, M. R. 1964. Further observations bearing on the
Biol. 35:206-220. bone-body fluid continuum: composition of the
Smith, H. M. 1947. Classification of bone. Turtox skeleton and serums of cyclostomes, elasmobranchs,
News 25:234-236. and bony vertebrates. Pages 151-180 in H. M. Frost,
Smith, H. W. 1961. From fish to philosopher. Double- ed., Bone biodynamics. Little, Brown and Co., Bos-
day, N.Y. ton.
Sprinz, R. 1965. A note on the mandibular intra- Urist, M. R. 1970. Induction and differentiation of
articular disc in the joints of Marsupialia and cartilage and bone cells. Pages 504-528 in O. A.
Monotremata. Proc. Zool. Soc. (London) 144:327- Schjeide and J. de Vellis, eds., Cell differentiation.
338. Van Nostrand Publishing Co., Amsterdam.
Tarlo, L. B. H. 1964. The origin of the bone. Pages Wake.D. B.,andR. Lawson. 1973. Developmental and
3-17 in H. J. J. Blackwood, ed., Bone and tooth. adult morphology of the vertebral column in the
Pergamon Press, Oxford. Plethodontid salamander Eurycea bislineata, with
Thompson, D'A. W. 1917. On growth and form. Cam- comments on vertebral evolution in the amphibia. J.
bridge Univ. Press, Cambridge. Morphol. 139:251-300.
Travis, D. F., C. J. Francois, L. C. Bonar, and M.J. Yasuda, Y. 1973. Differentiation of human limb buds
Glimcher. 1967. Comparative studies on the organic in vitro. Anat. Rec. 175:561-578.
matrices of invertebrate mineralized tissues. J. Ul- Zangerl, R. 1966. A new shark of the family Edestidae,
trastruct. Res. 18:519-550. Ornkhoprion hertwigi. Fieldiana Geol. 16:1-43.