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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. 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 membrane 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 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 understanding 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 Development Fund. The manuscript was critically read by Dr. E. T. Garside and by Dr. P. Person. biologist) to contribute the sort of papers 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 meaningful projections as to the evolutionary consequences resulting from the limitations (or potentials) of skeletal development 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 expression preserved in the fossil record) has not 329 330 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 tistaining 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 microscope (0rvig, 1951, 1957, 1965; Enlow the evolution of the vertebrate skeleton. and Brown, 1956; Moss, 19616; Halstead, 19696) and because chemical analyses can TYPES OF SKELETAL TISSUES now be carried out on both the inorganic I should perhaps define the skeletal tis- and the organic components of these tissues 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 intissues 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 unimportant, 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 specializausually preserved in the fossil record. tion, and their approximate times of apThe 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 jawless vertebrates), the earliest vertebrates for 1967, for a detailed classification). 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 the Cyclostomata are extinct. The members bone). of the extinct orders were all small (most 50 2) Cartilage: hyaline, elastic, fibrocartito 300 mm in length, some Osteostraci atlage, calcified, chondroid. taining 1 m in length) and are known cnly 3) Dentine: cellular and acellular. from fossilized cephalic shields. 4) Enamel. The notochord is assumed to have been 5) Tissues intermediate between the present as a supporting soft skeleton in all above. The basic cell type which produces these of these specimens. The presence or abmineralized tissues has been termed the sence of uncalcified cartilage can be argued Scleroblast by Moss (1964a), implying that only by inference (i) because cartilage, unthe tissues represent a family or race of like the notochord, is not a universal diagtissues 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 vertecrest). Thus, the osteoblast (mesodermal, ec- brates, especially whether it antedated the tomesenchymal) produces bone; the chon- appearance of bone, as it does in ontogenetdroblast (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 (Romer, 1942, 1963, 1964; Smith, 1947; duces enamel. 331 SKELETAL DIFFERENTIATION AND EVOLUTION 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) 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 mod1969a). 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 members of this group in association with a appearance. 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 calmal scales composed of acellular bone, the cified cartilage was also present. The earso-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. Ordovician Cambrian Pre Cambrian 1 Mammals Birds Note that acellular bone reappeared in teleosts. re U bo a a. < ,- re ichoiidr •p V peril c -a rm ha) W lage Amphibia Chondrichthyes Placoderms Osteichthyes Agnatha 0 X tine Reptila c idi Devonian Silurian re -5 mel Quaternary Cretaceous Jurassic Triassic Permian Pennsylvanian Mississipian £ V c CQ CO 332 BRIAN K. HALL FIG. 1. Bone containing globular structures (chondrocytes?) from (A) a cephalaspid, and (B,C) an antiarch. (From 0rvig, 1968.) in structure than the bone described previously. Some intermediate tissues were indeed 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 and calcified cartilage, in the early vertebrates. 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 osteocytes 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 between 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. enamel, and calcined cartilage in the Osteostraci. The replacement of cartilage by the formation of endochondral bone appears to have been a slightly later development 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 tissues which were, for example, bone, but bone that was simpler and more primitive 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 evolution of the teleosts from the actinopterygians, acellular bone was derived secondarily from cellular bone (Kolliker, 1859; Denison, 1963). The relationship of Agnathan aspidin to cellular bone has been more hotly debated, all possible views having been proposed: that the two evolved independently (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 333 SKELETAL DIFFERENTIATION AND EVOLUTION Hancox (1972). The fact that the two tissues appeared contemporaneously in the Ordovician in two distinct groups of Agnathans indicates that the two must have arisen earlier, either independently from less-specialized tissues or from a single primitive skeletal tissue. A further question which has been debated back and forth for decades is: "which of these skeletal tissues appeared first in the vertebrate skeleton during its evolution?" usually reduced to a question of "which came first, cartilage or bone?" This considerable debate was, to a large extent, fostered by the application of embryological theory to the study of skeletal evolution and illustrates the need for caution when making such applications. Because the vertebrate embryo was found to have a predominately cartilaginous skeleton, because cartilage precedes bone in endochondral bone formation, and because the cartilaginous fishes (the Chondrichthyes) were considered primitive, cartilage was assumed to have evolved before bone and phylogenies of the vertebrates were constructed accordingly (Fig. 2) (Romer 1942, 1963). However, as the data summarized in Table 2 indicate, enamel, dentine, calcified cartilage, and dermal bone (both cellular and acellular) share equal honors in terms of first appearance in the ancient verte- AMPHIBIA" Crossopterygii* Dipnoi* . 4 ^ . ^SARCOPTERYGir Jp Teleosts* ^ Actinopterygii* OSTEICHTHYES* Chondrichthyes^. .^. Cyclostomes PLACODERMS* QstArms' AGNATHA* FIG. 3. The evolution of the lower vertebrates assuming bone (*) appeared early in evolution. (Modified from Romer, 1964.) brates. Endochondral bone seems to have arisen later (first in the Middle Ordovician Osteostraci, then in the Silurian acanthodians, and in some of the placoderms) and to have come to prominence in those groups from which later groups evolved (the rhipidistian sarcopterygians which gave rise to the land vertebrates, and the actinopterygian fishes). Thus, the first vertebrate hard tissues were histologically diAMPHIBIA* verse and specialized. A phylogeny which takes these facts into account is shown in DIPNOI Figure 3. The question of the adaptive role played Crossopterygians* by the skeletal tissues in the early vertebrates has received considerable attention with the assumption that the factors reOSTEICHTHYES sponsible for the adaptations were the same factors as were responsible for the initial Placoderms" -^fcOstracoderms* evolution of the skeletal tissues. The following possible functions have been proposed: ^ CHONDRICHTHYES ^ Bone served a mechanical supporting function as it does today (Berrill, 1955; Schaeffer, 1961; Denison, 1963). The dermal skeleton served both as a reservoir and as a CYCLOSTOMATA permeability barrier to conserve calcium and especially to conserve phosphorus. FIG. 2. The evolution of the lower vertebrates assuming 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 mechanical support and for rapid growth, especially embryonic growth and development (Romer, 1942, 1963, 1964; Berrill, 1955; Denison, 1963). a restriction of the types of tissues produced 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." The transition from the reptiles to the mammals did involve fundamental changes in the associations and positions of skeletal elements, e.g., the evolution of the new jaw articulation (du Brul, 1964); the growth of the jaw (the sutural growth of the multiSKELETAL TISSUES DURING EVOLUTION 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 synrecent 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 fundamentetrapods. No advance in the structure of tally the same as were those of the Ordovibone was associated with the evolution of cian vertebrates. Thus, bone histology canthe reptiles from the amphibia or of the not be used to identify individuals of a particular species and often not members of mammals from the reptiles. Thus: the same class (Enlow, 1966). " . . .while much structural variation of bone tissue is found (throughout evolution), these differThe 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 differences in the structure of the components fossil vertebrates would appear to be as folthemselves" (Enlow and Brown, 1958, p. 212). lows: The skeletal tissues of the early OrAgain (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 verteis not possible to trace a precise series of progres- brates, although more intermediate tissues sive, increasingly complex developmental stages were present then than are present now. from ancient fish to modern animals." 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 tisFor 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 indoes not show a phylogenetic trend, but volved major changes in cell or tissue orindicates 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 considAs it can be maintained that skeletons of erable variation in the histological structure 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 day vertebrates, it seems reasonable to conclude 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 involve 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 later, that skeletal tissues are especially susceptible to modulations from environmental factors, then experimental studies on skeletal development and differentiation become extremely valuable as tools for understanding the evolution of the skeleton. This view has been aptly synopsized by Moss (1964a): " .. .intrinsic embryological phenomena, as modified by the extrinsic factors of mechanical function, forms the basis of a meaningful discussion of the evolution of skeletal tissue type." We might study the interaction between these two bodies of knowledge by examining available information on the developmental processes involved in producing the skeleton. This we shall do by following the life history of a "typical bone," an approach used previously in reviewing the histogenesis and morphogenesis of bone (Hall, 1971). We will ask a series of questions about skeletal development, present the current status of knowledge on that topic, and then determine whether the information is of any value in understanding aspects of the evolution of the skeleton. 335 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 migration of mesodermal cells into the lower jaw of the chick (Jacobson and Fell, 1941), or the migration of the ectomesenchymal cells from the neural crest into the skull, mandible, or pharynx (Johnson and Listgarten, 1972). The factors responsible for condensation are largely unknown, although cell adhesion may play a role (Ede, 1971; Moss, 1972ft). In the case of the ectomesenchymal cells of the neural crest which form the cartilage of the head, the stimulus for condensation results from interaction with the pharyngeal endoderm (Holtfreter, 1968). Epithelial-mesenchymal interactions The position of the condensed mesoderm or ectomesenchyme, and the shape, size, and rate of growth of the condensed cells, are determined by interaction with adjacent ectoderm (epithelia), the socalled epithelial-mesenchymal interactions. These interactions may involve the two-way interchange between a stationary layer of ectoderm and local mesoderm, as in the formation of the amniote limb, or they may involve establishment of an association between 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 capable of producing either cartilage or The first sign that skeletogenesis is immuscle up to Hamilton-Hamburger stage minent is condensation of mesodermal (or 24 (4-'/2 days of incubation). Thereafter ectomesenchymal) cells to form the anlage only the cells in the central condensed (primordium) of the bone. The position of mesoderm form cartilage and these are the the condensation within the embryo 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 additional skeletal tissue, indicating that the ectoderm influences the fate of the mesodermal 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 environmental factors stabilize the genome of cells in specific sites for skeletogenesis and further influence what type of skeletal tissue will form. Teeth form at the junction between the buccal epithelium and those ectomesenchymal 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 adjacent 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 auditory meatus, and their associated tissues, involve epithelial-mesenchymal interactions 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 interactions involved in the formation of the tooth. Double arrows indicate inductive interactions. (Modified from Koch, 1972.) mesenchyme. For example, the mesenchyme 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 (ectomesenchyme?) which shifts in position. Cells destined to form cartilage, bone, or muscle arise in three separate mesenchymal centers outside the mandible (each associated with a "transitory epithelial thickening") and then migrate into the mandible where they differentiate (Jacobson 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 during evolution? Two obvious areas to consider are the origin of the jaws during evolution of the gnathostomes and the evolution of the mammalian jaw articulation, both of which involve the shifting in position, and modification of function, of pre-existing skeletal elements. In the evolution of the gnathostome vertebrates 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 cartilage), also probably of neural crest origin, and a number of membrane bones, to be discussed later. The simplest developmental 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 development of membrane bones, I will turn to the evolution of the mammalian jaw articulation. The reptilian jaw articulates through the articular of the lower jaw and the quadrate of the upper jaw. In the transition to the mammals these two endochondral bones migrated posteriorly to form, respectively, the malleus and the incus of the middle ear. A new jaw articulation developed between a newly formed condyle of the now greatly expanded dentary (which comprises the lower jaw) and the squamosal of the skull. Ample evidence illustrating 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 inGnathostome. First gill arch modified as jaws. C, Os- volves consideration of the skull as a functeichthyes. Jaws braced by second gill arch. (From du tional unit and the changing conformation Brul, 1964.) 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 fundamenrod 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 memappears 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 mesencould 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 memplane 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 cartilage models of long bones. In addition to Meckel's cartilage and an The intramembraneous (perichondral, endochondral bone(s) which develops in 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 chondrocytes are in turn either replaced by, or converted into, endochondral bone (Holtrop, 1966; Crelin and Koch, 1967; Hall, 1970). A similar situation has been postulated for the initiation of membrane bone formation around Meckel's cartilage, although 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 ossification. One could suggest that the membrane bones surrounding Meckel's cartilage (and this is a topographic arrangement not duplicated elsewhere in the body) were originally 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 subsequently 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 published an extensive series of observations indicating that Meckel's cartilage is completely replaced by endochondral bone in some mammalian species, incompletely replaced in others, and that in some it remains as a cartilaginous rod, the chondrocytes failing to hypertrophy. In those where replacement is partial, bone forms only adjacent to hypertrophicchondrocytes. Other areas of the cartilage fail to undergo hypertrophy and remain cartilaginous. 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 embryonic 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 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 condensation 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 ossification, whereas neural influences alone determine intramembranous ossification? The somatic mesoderm in which the vertebrae develop also requires an induction from the notochord before chondrogenesis can commence (Lash, 1968). As in the development of the endochondral bones of the skull, the endochondral vertebrae receive a dual inductive stimulus both from the notochord and from the spinal cord. In fact in some vertebrates, e.g., the salaman- 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 6. The inductive interactions between brain, 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.) 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 timing of skeletogenesis depends primarily on ectodermal-mesodermal interactions, a concept which can be very usefully applied to the study of the evolution of skeletal tissues 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 following types of scleroblasts will form: chondroblast, chondrocyte (cartilage); osteoblast, osteocyte (bone); fibroblast (ligament, tendon); odontoblast (dentine); ameloblast (enamel). Each is characterized by a particular extracellular product and by a particular structure. Along with the concept that any mesenchymal cell has the potential to become a scleroblast given the appropriate stimulus is the concept that the various types of scleroblasts are members of an interrelated and potentially interconvertible family (Moss, 1964a, 1968c, 1969; Hall, 1970, 1971). The existence, in both recent and fossil vertebrates, of tissues intermediate 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 experimental manipulation provides the empirical verification of the concept. Thus, to endeavor 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 common 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 concept 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 mesenchymal in appearance (hence the name ectomesenchyme) and migrate to various sites within the embryo. In the Amphibia, the chondroblasts, osteoblasts, and odontoblasts of the cranial skeleton all arise from the neural crest (de Beer, 1958). Experimental studies on the chick embryo have provided similar conclusions for the origin of cranial chondroblasts and several experiments 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 cartilage) in the cartilages of the head and in the visceral arches. Although labeled bone was not observed (and osteogenesis is well advanced in the head of a 9-day embryo), labeled undifferentiated mesenchymal cells were seen and were thought to be osteogenic precursors. Hammond and Yntema (1964) removed neural crest without replacement from similarly aged embryos and noted subsequent 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; origin of bone from similar cells is based on inferential evidence (further evidence is being sought in my laboratory). Jollie (1971) has studied the development of the head of the embryonic shark, Squalus, and has shown that Meckel's cartilage and the teeth (both of which are derived 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 Jolsues 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 consethe 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 possition 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 verteern 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 orwhere it is acellular (Moss, 1970), and in gan, ideally suited to evolutionary modHeptanchus (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 The previous studies provide experibases of which were imbedded in bone. 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 succeeddevelopment 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 remain at the surface to become the dermal too restrictive in concept. exoskeleton (Tarlo, 1964; Moss, 19686). Much of the skeleton of the early verte- Hay (1964) has shown that collagen, probrates 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 proneural 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 underbrate 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 ectoderm, and modulation to different This discussion of the origin of skeletal scleroblast types equally early. The de- tissues from the neural crest raises the quesvelopment 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 anecdote on this matter—about the Professor who delayed publication of his student's research 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 Matsumura (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 collagen (Moss, 1963, 1970; Mossetal., 1964), or the same (Matsumura, 1972). Various invertebrates (e.g., squids, tunicates, and Branchiostoma) form true epidermal cartilage, and some produce composite musculoskeletal tissues as in the intimate association of muscle and cartilage in the odontophoral cartilage of grazing gastropods (Person and Philpott, I969a,b; Carriker et al., 1972). Thus, not only can ectodermal cells produce 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 question of the origin of vertebrate skeletal tissues during the evolution of the vertebrates from an invertebrate stock. INVERTEBRATE ORIGINS OF THE VERTEBRATE SKELETON There is an essential unity in mineralization 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 vertebrate 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 mechanisms, but the selection and modification of 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). Calcification in both vertebrates and invertebrates 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 ectodermal (mollusc shells) or mesodermal (echinoderm skeletons) origin but have not evolved mechanisms of interactions between the two germ layers. Moss (1968c) has enumerated four processes 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 combined (ecto-mesodermal) calcified tissue with active participation by the ectoderm; (ii) induction between the mesoderm or ectomesenchyme and the ectoderm; (iii) delamination. The acquisition of the hard skeleton by the first vertebrates potentially could have occurred quite rapidly for the scleroblasts respond very quickly to functional demand. EPIGENETIC REGULATION OF SCLEROBLAST ACTIVITY Recent studies on the formation of secondary cartilage on the membrane bones of the avian skull will serve to illustrate one environmental factor which can drastically alter scleroblast behavior and tissue differentiation. The environmental factor involved 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, cartilage arises on membrane bones at their articular surfaces late in the 10th or early in the 11th day of incubation. This coincides with the time of increased overall movement by the embryo (Hamburger et al., 1965). If the embryos are paralyzed, or if the membrane bones are cultured in a stationary culture, cartilage fails to form (Hall, 1967, 1968, 1972; Murray and Smiles, 1965). If mechanical stress is applied to these bones in culture, cartilage can be induced, the amount of cartilage varying in proportion to the duration of the stimulus (Hall, 1968). The interesting finding is that the cartilage which forms in the 1 lth day, and the bone which was forming earlier and which continues to form after 11 days, arise from a common pool of mesenchymal cells. Where the cells are protected from the mechanical stimulus osteogenesis continues. Where the cells are mechanically stressed they become chondrogenic, and they can, if the stress is intermittently applied and removed, alternate between the two scleroblast (Chondroor Osteoblasts) types (Murray and Smiles, 1965). Alterations in mechanical stresses were extremely important in the evolution of the jaws and in the evolution of the entire vertebrate head and, as these recent experiments indicate, mechanical stress is a potent modulator of scleroblast behavior. 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 intramembraneous 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 characteristic of the species arise in bone disease, such as osteosarcoma or chondrosarcoma. Bone forms in "cartilaginous" fishes. The primitive scleroblast (both ontogenetically and phylogenetically) is an extremely adaptive cell with perhaps more hidden potential than most other cell types. A study of the skeletal tissues of the ancient vertebrates which neglects such modifying epigenetic factors is at the very least incomplete, and at worst inaccurate. Cellular and acellular bone The views on the relationships between cellular and acellular Agnathan bone discussed earlier take on a new dimension when viewed in the light of the development of the acellular bone of modern higher teleosts. Such acellular bone is found in both freshwater and in saltwater species and so appears not to be correlated with retention of calcium or of phosphorous (Moss, 1961a, 1965). The functional Moss (1969) has shown that, within the significance of acellular bone remains unreptilian 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 ostendon, 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 ostential to produce the whole range of teogenesis by metaplasia of secondary carskeletal 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 tions of cytoplasm into supporting tissue matrix are seen in some invertebrate cartilages 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 evolution of acellular bone (aspidin) from cellular dermal bone we should really be considering the origin of cellular bone from cellular bony or other connective tissues. The important evolutionary question is then really a developmental question: Why do the osteocytes not persist? (0rvig, 1957,1965; Calder and Hall, unpublished). Sesamoids and secondary centers of ossification 343 ossification within long bones likewise involved progressive adaptation of preexisting mechanisms of chondro- and osteogenesis, 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 differentiative potential of the constitutent cells of the skeleton. To summarize the answer to the question of how the various skeletal tissue types form at specific sites, it appears that the specificity comes from local environmental factors such as contact with specific epithelia, local mechanical stresses, and not always from intrinsic predetermination of the potentially skeletogenic cells. (The cells are biased toward skeletogenesis, but epigenetic factors determine which particular scleroblast type will develop at a given site.) These cells are then potentially and actually interconvertible and the consequences of such interactions and modulations must be kept in mind when considering the evolution of skeletal tissues and especially when basing phylogeny on tissue and cell structure. Some structures which are almost universally present in the long bones of recent mammals, such as secondary centers of ossification within the epiphyses, or sesamoid bones at joints, appear to have arisen with the mammals in the Jurassic (Haines, 1969). Do these not indicate the progressive evolution of skeletal tissue structure? Apparently not, if we look at the development SIZE AND SHAPE OF THE SKELETON of such structures. Sesamoid bones are known to be intratendinous ossifications We may now turn to the question: What (Barnet and Lewis, 1958) or intradetermines the size and shape of the skeleligamentous ossifications (Burton, 1973) ton? and it is known that the ornithischian dinosaurs possessed ossified tendons Morphogenesis of the skeleton (Haines, 1969) and that reptiles tend to show tendinous rather than periosteal osOnce 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 contive 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 conthe 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 decally 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. A study of the determination of the mor(the extra stresses imposed on the joints by phology of particular bones within the the adoption of terrestrial life). skeleton sheds considerable light on the relThe 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 maintenance of form in the animal and plant kingdoms as an adaptation to the environment but avoided the question of the relationship between ontogenetic adaptation and the inheritance of form and pattern. Murray (1936) summarized the early literature 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 acting 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 morphology, accompanied by considerable linear growth) of a skeletal element is independent of functional demands, and is under genetic control (Howell, 1917; Felts, 1961; Chalmers and Ray, 1962; Mawdsley and Ainsworth Harrison, 1963). For example, if the mesodermal primordium of a bone, or the early bony anlage after initiation of osteogenesis or chondrogenesis, is grafted to the chorio-allantoic membrane, or transplanted subcutaneously or intramuscularly, 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 normally (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 hypertrophy, and amounts of intercellular matrix produced per cell. Once this fundamental form is established, the development of "minor" architectural features of the bone (ridges for attachment of muscles, ligaments etc.) depends 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 continued 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 processes 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 minor elements of the bone's form could occur quite rapidly as ontogenetic modifications 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 interactions) whether the cells destined to form the tissues of the skeleton will differentiate 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, provided that only the minor features of the skeleton are included. Some examples from recent experimental studies are provided below. If the vitreous humor is drained from the eye(s) of the embryonic chick late in the 4th day of incubation, microphthalmia is induced 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. Furthermore, 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 morphogenesis, growth, and pattern formation of the adjacent and subjacent skeletal ele- SKELETAL DIFFERENTIATION AND EVOLUTION merits which form parts of the same functional unit. The muscles of the head also play a role in controlling the growth of this functional unit. Bilateral masseterectomy in the newborn 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). However, the basic architectural features of the bones are unaffected by surgical manipulations to the adjacent soft tissues (Pratt, 1943). The types of tissues produced at a given site within the skeleton, the association between adjacent skeletal units, and the plasticity of skeletal tissues for developmental modulations and responses to environmental stimulii during evolution are amply illustrated by the following studies. Bock (196CM) and Bock and Morioka (1968) have carried out a series of studies, illustrating the repeated evolution of elements of the avian skull. For example, the palatine process of the premaxilla may be either fused or unfused and has been lost and reappeared many times during avian evolution (presumably in response to newly appearing stresses and pressures). The median process of the mandible has likewise appeared where stress on the mandible and strength of the quadrate dictated (Bock, 19606), a situation which can be induced experimentally by paralysis of avian embryos (Murray and Drachman, 1969). Simonetta (1960) and Bock (1964) have reviewed the question of the evolution of the kinetic avian skull and the factors which modify its skeletal elements of the skull. 345 viously unconnected skeletal elements as described above often leads to the development of a new articulation, or to the modification of existing sutures, joints, or articulations to accommodate the evolution of the complex musculo-skeletalconnective tissue functional unit. The basic (primitive?) suture between two adjacent bones is what has been termed the flat suture (Moss, 1957) and consists of two bone surfaces opposed to one another without interdigitation. Such sutures may be modified in response to the functional demand made on them and develop interdigitations, overlapping surfaces, etc. Such is the case in the skull of the woodpecker and provides an answer to the enigmatic question: "Why don't woodpeckers get headaches?" It turns out that the suture between the frontal and the nasal bones is an overlapping one, enabling one bone to ride over the other and absorb some of the stresses which would otherwise be directed onto the articulation. This implies changes in the soft tissues associated with the articulations related to functional demand, and although evidence is difficult to obtain from the fossil record, numerous experimental examples are available: for example, the presence or absence of, and the degree of fibrous development in, the intra-articular discs of the temporo-mandibular joints of Marsupials and Monotremes (Sprinz, 1965), or the transformation of fibrous articular tissues to fibrocartilage to allow the human temporo-mandibular joint to adapt morphologically to mechanical stresses (Moffett et al., 1964). What are the implications of these studies for establishment of phylogenetic trends in skeletal tissues? Single character analyses such as those of 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 between closely related species and highlight These studies illustrate the difficulties inthat there are many ways by which skeletal volved in establishing a phylogeny of elements may respond to environmental skeletal tissues based on the genetic selecconditions. The final form of the character tion of progressively more well-adapted tisdepends 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 hisfrom 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 evirial), length of the development period, age dence, in the form of tissues intermediate at maturity, seasonal feeding cycles, and between those recognized as discrete endpoints (bone, cartilage, dentine) that their hormonal milieu. When we say that no major structural cells were highly adaptive even at the outset 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 conadvancement 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 tishas 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. Contypes; decrease in the amount of bone per vergence and divergence ought to be exanimal; decrease in the number of bones pected and according to Jollie (1968) ocper 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. Modthing they reflect dimunition of skeletoge- ulation to bone facilitated storage of essennesis 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 recent vertebrates form a coherent, interrelated class, according to a number of criteria: embryological development, proREFERENCES duction of extracellular matrices consisting of a fibrous protein and a carbohydrate Alexander, R. McN. 1975. Evolution of integrated design. Amer. Zool. 15:419-425. component, ability to calcify the matrix, the S. P. 1967. A survey of shark hard parts. potential to modulate to other cell types, Applegate, Pages 37-67 in P. W. Gilbert, R. F. Mathewson, and high degree of responsiveness to modificaD. P. Rail, eds., Sharks, skates and rays. Johns Hoption by environmental factors. There is, kins Press, Baltimore. therefore, justification in speaking of Barnett, C. H., and O. J. Lewis. 1958. The evolution of some traction epiphyses in birds and mammals. J. skeletal tissues as closely related to one 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 American blackbirds. Auk 68:411-440. the very high degree of plasticity shown by these cells, their readiness to modulate in Beer, G. de. 1958. Embryos and ancestors. 3rd ed. Oxford Univ. Press, London. response to epigenetic factors, and their Berrill, N.J. 1955. The origin of the vertebrates. Oxearly appearance as specialized tissues in ford Univ. Press, London. SKELETAL DIFFERENTIATION AND EVOLUTION 347 54:803-822. Biltz, R. M., and E. D. Pellagrino. 1969. The chemical anatomy of bone. 1. A comparative study of bone Enlow, D. H. 1969. The bone of reptiles. Pages 45-80 in C. Cans, ed., Biology of the Reptilia. Vol. 1. composition in sixteen vertebrates. J. Bone Joint Academic Press, New York. Surg. 51A:456-466 Bock, W. J. 1960a. The palatine process of the pre- Enlow, D. H., and S. O. Brown. 1956. A comparative histological study of fossil and recent bone tissues. maxilla in the Passeres. A study of the variation, Part I. Texas J. Sci. 8:405-443. function, evolution and taxonomic value of a single character throughout an avian order. Bull. Mus. Enlow, D. H., and S. O. Brown. 1957. A comparative histological study of fossil and recent bone tissues. Comp. Zool. 122:361-488. Part II. Texas J. Sci. 9:186-214. Bock, W. J. 19606. Secondary articulation of the avian mandible. Auk 77:19-55. Enlow, D. H. and S. O. Brown. 1958. A comparative histological study of fossil and recent bone tissues. Bock, W. J. 1964. Kinetics of the avian skull. J. MorPart III. Texas J. Sci. 10:187-230. phol. 114:1-42. Bock, W. J., and H Morioka. 1968. The ecthemoid- Felts, W. J. L. 1961. In vivo implantation as a technique in skeletal biology. Int. Rev. Cytol. 12:243-302. mandibular articulation in some Meliphagidae (Aves). Amer. Zool. 8:808. (Abstr.) Felts, W. J. L., and F. A. Spurrell. 1966. Some structural and developmental characteristics of cetacean Bock, W. J., and G. von Wahlert. 1965. Adaptation (Odontocete) radii. A study of adaptive osand the form-function complex. Evolution 19:269teogenesis. Amer. J. Anat. 118:103-134. 299. Brul, E. L. du. 1964. Evolution of the temporoman- Friant, M. 1959. Sur l'ossification enchondrale du cardibular joint. Pages 3-27 in B. G. Sarnat, ed., The tilage de Meckel chez les Rongeurs. Bull. GR. Int. temporomandibular joint. C. C. Thomas, SpringRes. Sci. Stomatol. 4:1-11. field, 111. Friant, M. 1964. Sur l'ossification du cartilage de MecBurton, P. J. K. 1973. Structure of the depressor mankel d'une chauvre-souris, le grand Murin (Chiropdibulae 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. Dal22:253-275. 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 in mice. J. Dent. Res. 50:1260-1267. Chalmers, J., and R. D. Ray. 1962. Transplantation 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. A study of inherited skeletal disorders in animals. Cracraft, J. 1968. The lacrimal-ectethmoid bone comBlackwell Scientific Publications, Oxford. plex in birds: a single character analysis. Amer. Midland Natur. 80:316-359. Haines, R. W. 1969. Epiphyses and sesamoids. Pages Crelin, E. S., and W. E. Koch. 1967. An autoradiogra81-116 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 cartibrate calcified skeleton. Clin. Orthop. Related Res. lage by membrane bones under the influence of 31:141-152. mechanical stimulation applied in vitro. Life Sci. 6:663-667. Drachman, D. B., and L. Sokoloff. 1966. The role of 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 tissues. Biol. Rev. Camb. Philos. Soc. 45:455-484. Enlow, D. H. 1966. An evaluation of the use of bone 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 transSpringer-Verlag, Heidelberg. formation into bone in the embryonic chick. Anat. Enlow, D. H. 1968. Wolffs law and the factor of arRec. 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 spinal chick embryos between 8 and 17 days of incubation. 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 protein 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 inductive and morphogenetic processes. Pages l-30m R. Fleischmajer, ed., Epithelia-mesenchymal interactions. Williams and Wilkins, Baltimore. Holtrop, M. E. 1966. The origin of bone cells in endochondral 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 effects of stress and strain on bone development. Anat. Rec. 13:233-252. Jacobson, W., and H. B. Fell. 1941. The developmental 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 principle 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. Observations on the migration, interaction, and early differentiation of orofacial tissues. Pages 55-80 in H. C. Slavkin and L. A. Bavetta, eds., Developmental aspects 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. 0rvig, 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 calcified 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 microstructure 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 formation des arcs branchiaux et du squelette visceral. J. Embryol. Exp. Morphol. 31:453-477. McLean, F. C, and M. R. Urist. 1968. Bone. Fundamentals 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 interactions as the basis for morphological change in evolution. Araer. Zool. 15:315-327. Mereel, M. 1967. Recherches sur la relation inductrice entre chondrocytes et perioste dans le tibia erabryonnaire 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. Environmental factors determining the growth and development 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. Transplantation studies of factors in skeletal organogenesis. II The response of the immature mouse humerus to longitudinal compressive forces. Amer. J. Phys. Anthropol. 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 variation. Acta Anat. 46:343-362. Moss, M. L. 19616. The initial phylogenetic appearance 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 vertebrate dermal bone and teeth. I. The epidermal coparticipation 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. Linnean Soc. N.S.W. 51:187-263. Murray, P. D. F. 1928. Chorio-allantoic grafts of fragments 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 related 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 remarks 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. 16:551556. 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 chemical organization of teeth. Academic Press, New York. 0rvig, T. 1968. The dermal skeleton: general considerations. 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 cartilages. Ann. N. Y. Acad. Sci. 109:113-126. Person, P., and D. E. Philpott. 1969a. The nature and significance of invertebrate cartilages. Biol. Rev. Camb. Philos. Soc. 44:1-16. Person, P., and D. E. Philpott. 19696. The biology of cartilage. I. Invertebrate cartilages: Limulus gill cartilage. 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 cartilage. 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 observations 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 adaptation. 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 animalsomatic 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 feeding mechanism. Amer. Zool. 1:187-204. Schowing, J. 1968a. Influence inductrice de I'encephale 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'encephale 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. Embryol. Exp. Morphol. 19:23-32. Schowing, J. 1968c. Influence inductrice de l'encephale embryonnaire sur le developpement du crane chez le poulet. III. Mise en evidence du role 350 BRIAN K. HALL inducteur de l'encephale dans l'osteogenese du Urist, M. R. 1962. The bone-body fluid continuum: calcium and phosphorous in the skeleton and blood crane embryonnaire du poulet. J. Embryol. Exp. of extinct and living vertebrates. Perspect. Biol. Morphol. 19:83-94. Med. 6:75-115. Sicher, H. 1966. Orban's oral histology and embryology. 6th ed. C. V. Mosby Co., St. Louis. Urist, M. R. 1963. The regulation of calcium and other ions in the serums of hagfish and lampreys. Ann. N. Simonetta, A. M. 1960. On the mechanical implicaY. Acad. Sci. 109:294-311. tions of the avian skull and their bearing on the 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 skeleton and serums of cyclostomes, elasmobranchs, Smith, H. M. 1947. Classification of bone. Turtox and bony vertebrates. Pages 151-180 in H. M. Frost, News 25:234-236. ed., Bone biodynamics. Little, Brown and Co., BosSmith, H. W. 1961. From fish to philosopher. Doubleton. day, N.Y. 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) 144:327Schjeide 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 comments on vertebral evolution in the amphibia. J. Thompson, D'A. W. 1917. On growth and form. CamMorphol. 139:251-300. bridge Univ. Press, Cambridge. Travis, D. F., C. J. Francois, L. C. Bonar, and M.J. Yasuda, Y. 1973. Differentiation of human limb buds in vitro. Anat. Rec. 175:561-578. Glimcher. 1967. Comparative studies on the organic 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.