Evolutionary Consequences of Skeletal Differentiation

Transcription

1 AMER. ZOOL., 15: (1975). Evolutionary Consequences of Skeletal Differentiation BRIAN K. HALL Department of Biology, 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. 329 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

2 330 BRIAN K. HALL markedly changed with time. If these hold true we may justifiably begin to examine experimental results pertaining to skeletal differentiation and plasticity and apply these to interpretations of the evolution of the vertebrate skeleton. TYPES OF SKELETAL TISSUES I should perhaps define the skeletal tissues which will be discussed, for I shall be concerned primarily with the hard (mineralized) tissues of the skeleton. Other tissues of the musculo-skeletal system, such as ligaments, tendons, fibrous and other connective tissues, vascular, muscular, and hemopoietic elements, will take second place in the discussion. This will be done, not because these other tissues are unimportant, for they obviously are important, especially in the context of the evolution of complex, multi-tissue functional units (see Bock and von Wahlert, 1965; Moss, 1968<2; Alexander, 1975), but because they are not usually preserved in the fossil record. The mineralized skeletal tissues may be broadly classified into four types (see 0rvig, 1967, for a detailed classification). 1) Bone: intramembraneous and endochondral, cellular and acellular, cancellous and lamellar (see Smith, 1947, for an excellent summary of the classification of bone). 2) Cartilage: hyaline, elastic, fibrocartilage, calcified, chondroid. 3) Dentine: cellular and acellular. 4) Enamel. 5) Tissues intermediate between the above. The basic cell type which produces these mineralized tissues has been termed the Scleroblast by Moss (1964a), implying that the tissues represent a family or race of tissues with close affinities one to another. Such cells may be mesodermal, ectodermal, or ectomesenchymal (from the neural crest). Thus, the osteoblast (mesodermal, ectomesenchymal) produces bone; the chondroblast (mesodermal, ectomesenchymal ectodermal? ) produces cartilage; the Odontoblast (mesodermal) produces dentine; and the ameloblast (ectodermal) produces enamel. The possibility of correlating recent and fossil skeletal tissues has recently been greatly enhanced, because fossil skeletal tissues can now be decalcified and sectioned, both for the light and for the electron microscope (0rvig, 1951, 1957, 1965; Enlow and Brown, 1956; Moss, 19616; Halstead, 19696) and because chemical analyses can now be carried out on both the inorganic and the organic components of these tissues (Mathews, 1966, 1967; Ho, 1967; Biltz and Pellegrino, 1969; Kobayashi, 1971; Hancox, 1972). The latter studies have indicated essential similarity in the chemistry of the skeletal tissues of fossil and recent vertebrates. SKELETAL TISSUES OF ORDOVICI AN VERTEBRATES A brief review of the skeletal tissues of the early vertebrates will serve to illustrate their homology with the tissues of the present-day vertebrates, their general complexity and high degree of specialization, and their approximate times of appearance in the fossil record. I will consider the members of the Class Agnatha (the jawless vertebrates), the earliest vertebrates for which fossil skeletal material is available. The major orders within the Agnatha and their distribution within the geological time scale are summarized in Table 1. All except the Cyclostomata are extinct. The members of the extinct orders were all small (most 50 to 300 mm in length, some Osteostraci attaining 1 m in length) and are known cnly from fossilized cephalic shields. The notochord is assumed to have been present as a supporting soft skeleton in all of these specimens. The presence or absence of uncalcified cartilage can be argued only by inference (i) because cartilage, unlike the notochord, is not a universal diagnostic vertebrate tissue, and (ii) because if uncalcified it is unfossilizable. Its inferred presence or absence in the early vertebrates, especially whether it antedated the appearance of bone, as it does in ontogenetic endochondral ossification, has led to numerous ingenious but speculative theories of vertebrate evolution, adaptive radiation, and the origin of skeletal tissues (Romer, 1942, 1963, 1964; Smith, 1947;

3 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) Order: Heterostraci (early ordovician to late devonian) Coelolepida (middle ordovician to late devonian) Berrill, 1955; Jarvik. 1959; Urist, 1962; Denison, 1963; Moss, 1961*, 1964a, 1968o,c; 0rvig, 1967, 1968; Halstead, 1969a). The skeletal tissues which have been preserved in the fossil record are summarized in Table 2 along with their time of earliest appearance. The oldest vertebrates known (Archodus, Palaeodus), species of unknown affinity from the early Ordovician of Russia, possessed a dermal skeleton which consisted of isolated denticles composed of dentine (Denison, 1963). The Anaspida (Anaspis, Birkenia, Pterolepis) possessed isolated dermal scales composed of acellular bone, the so-called aspidin(e). The Heterostraci (Astraspis, Pycnaspis, Eriptychius), from the early and middle Ordovician, had a solid dermal armor of acellular, coarse-fibered aspidin, covered by a layer of dentine which in some was in turn covered by a layer of enamel (enameloid) (Denison, 1963; Halstead, 1969ft; 0rvig, 1951). In one species, Eriptychius, cartilage exhibiting spherulitic (globular, calcospheritic) calcification has been observed. Similar cartilage is seen in the mandible and palatoquadrate of modern sharks (Applegate, 1967) and has been observed in homografted hyaline cartilage in man (MacConaill, 1973). Perichondral bone was also present in some of the members of this group in association with a neurocranium presumed to have consisted of uncalcified cartilage. The Osteostraci (Alaspis, Cephalaspis, Hemicyclaspis) had a dermal exoskeleton of cellular bone capped by dentine and enamel, and a pharyngeal and cranial endoskeleton of endochondral bone (Fig. 1). Spherulitic and globular calcified cartilage was also present. The earliest jawed vertebrates, the Acanthodii (middle Silurian to Permian) possessed a similar range of tissues, except that their bone was always cellular. Thus, during the early Ordovician two major combinations of skeletal tissues were present within the Agnatha: acellular bone, dentine, enamel, and calcified cartilage in the Heterostraci, and cellular bone, dentine, TABLE 2. A summary of the occurrence of mineralized skeletal-tissues in the vertebrate fossil record. First appearance of major groups (classes) is also shown. Quaternary Cretaceous Jurassic Triassic Permian Pennsylvanian Mississipian Devonian Silurian Ordovician Cambrian Pre Cambrian Mammals Birds Reptila Amphibia Chondrichthyes Placoderms Osteichthyes Agnatha mel W tine c V a lage p U ha) re bo idi a. < re -5 c0 X peril,- re rm V c CQ ichoiidr -a CO 1 Note that acellular bone reappeared in teleosts.

4 332 BRIAN K. HALL FIG. 1. Bone containing globular structures (chondrocytes?) from (A) a cephalaspid, and (B,C) an antiarch. (From 0rvig, 1968.) 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 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. 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

5 SKELETAL DIFFERENTIATION AND EVOLUTION 333 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* DIPNOI OSTEICHTHYES Crossopterygians* Placoderms" -^fc- Ostracoderms* ^ CHONDRICHTHYES ^ CYCLOSTOMATA FIG. 2. The evolution of the lower vertebrates assuming bone (*) appeared late and off the main evolutionary line. (Modified from Romer, 1964.) AMPHIBIA" Crossopterygii* Dipnoi*.4^. Teleosts* ^SARCOPTERYGir Jp ^ 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 diverse and specialized. A phylogeny which takes these facts into account is shown in Figure 3. The question of the adaptive role played by the skeletal tissues in the early vertebrates has received considerable attention with the assumption that the factors responsible for the adaptations were the same factors as were responsible for the initial evolution of the skeletal tissues. The following possible functions have been proposed: 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 permeability barrier to conserve calcium and especially to conserve phosphorus. The ultimobranchial gland and, in later groups, the parathyroid gland aided in this

6 334 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). SKELETAL TISSUES DURING EVOLUTION Enlow and Brown (1956, 1957, 1958) have provided a very detailed comparative survey of the histology of both fossil and recent bone, and more recently Enlow (1969) has reviewed the bone of the reptiles and Ricqles (1968, 1969, 1972) that of the tetrapods. No advance in the structure of bone was associated with the evolution of the reptiles from the amphibia or of the mammals from the reptiles. Thus: "...while much structural variation of bone tissue is found (throughout evolution), these differences usually involve variation in the arrangements of fundamental components and not major differences in the structure of the components themselves" (Enlow and Brown, 1958, p. 212). Again (p. 220) "...in the history of bone tissue, a single evolutionary line cannot be recognized. It is not possible to trace a precise series of progressive, increasingly complex developmental stages from ancient fish to modern animals." Again: "Anatomical specializations are not per se necessarily correlated with phyletic modifications in the histological structure of hard tissues" (0rvig, 1965, p. 554). For example the degree of mineralization of the skeleton, as indicated by its density, does not show a phylogenetic trend, but indicates local adaptation to environmental conditions such as mechanical stress, equilibrium, buoyancy, protection, temperature, conservation of energy, as has been documented for the Ostracoderms (Schaeffer, 1961) and for the Cetacea (Felts and Spurrell, 1966). There is very considerable variation in the histological structure of fish bone and cartilage (explosive radiation) which is not seen in the Amphibia or the Reptilia (Moss, 1964a, 1969), indicating 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 multiboned reptilian mandible vs. the apical growth of the mammalian dentary Sicher, 1966); the attachment of the teeth to the jaw (ankylosed in reptiles and a syndesmosis in mammals). These involved changes in the developmental processes, but the tissues produced were fundamentally the same as were those of the Ordovician vertebrates. Thus, bone histology cannot be used to identify individuals of a particular species and often not members of the same class (Enlow, 1966). The conclusions to be drawn from an examination of the histology of the bones of fossil vertebrates would appear to be as follows: The skeletal tissues of the early Ordovician vertebrates were essentially the same as are those of present-day vertebrates, although more intermediate tissues were present then than are present now. The first mineralized vertebrate tissues known from the fossil record were complex and specialized (implying earlier unfossilized and perhaps simpler (?) skeletal tissues). Skeletal evolution in the vertebrates, at least since the Ordovician, has not involved major changes in cell or tissue organization but rather has involved adaptive responses of already specialized and plastic tissues to new local environmental changes. THE BORDERLAND BETWEEN EMBRYOLOGY AND PALAEONTOLOGY As it can be maintained that skeletons of the earliest vertebrates were highly specialized structures whose tissues were as complex (advanced) as are those of present-

7 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. INITIATION OF SKELETOGEXESIS The first question is a double-barreled one: How does the development of a bone commence, and what determines where that bone will develop? Mesenchymal condensation The first sign that skeletogenesis is imminent is condensation of mesodermal (or ectomesenchymal) cells to form the anlage (primordium) of the bone. The position of the condensation within the embryo defines the subsequent position of the bone within the adult; the shape of the condensation defines the future basic shape of the SKELETAL DIFFERENTIATION AXD EVOLUTION 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 to these specialized epithelia, but outside the condensation, can form skeletal tissues, but only the tissues within the condensation do form skeletal tissues (reviewed by Hall, 1971). A good example is the formation of the cartilaginous primordia of the long bones in the limb of. the embryonic chick. Any mesodermal cell in the limb bud is capable of producing either cartilage or muscle up to Hamilton-Hamburger stage 24 (4-'/2 days of incubation). Thereafter only the cells in the central condensed mesoderm form cartilage and these are the cells most closely associated with the specialized ridge of ectoderm the apical ectodermal ridge. An extra ridge grafted to

8 336 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 neural tube PHARYNGEAL ENDODERM ECTODERM neural crest=buccal epithelium 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

9 SKELETAL DIFFERENTIATION AND EVOLUTION 337 FIG. 5. The origin of the jaws, /f.jawless Agnatha.B, Gnathostome. First gill arch modified as jaws. C, Osteichthyes. Jaws braced by second gill arch. (From du Brul, 1964.) associated specific epithelium, and producing in the new site, a cartilaginous and bony rod as it would have done in the gill arch. The alternatives of establishment of a new pathway of migration of neural crest directly from the neural tube to the anterior segment of the lower jaw and of subsequent establishment of new site and tissue-specific epithelia to induce the now adjacent mesenchyme to produce Meckel's cartilage appears less likely (i) because several developmental processes are involved, and (ii) because the pre-existing cartilagedetermined tissues within the gill arch could be transposed more readily. However, in the avian embryo, neural crest cells do follow such a route along a cleavage plane between the ectoderm or pharyngeal endoderm and the adjacent mesoderm (Johnson and Listgarten, 1972). In addition to Meckel's cartilage and an endochondral bone(s) which develops in Meckel's cartilage (see below), the lower jaw contains a number of membrane bones 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 stimulus which initiated the movements involves consideration of the skull as a functional unit and the changing conformation of the skull base, changes in muscle mass, new directions of stress (see below and also Noble, 1973). It may be that no fundamental changes in the genome were required to bring about the development of this new articulation. The origin of membrane bones The question of the origin of the membrane bones of the lower jaw, or indeed, of membrane bones in general, irrespective of their location, is one to which we shall now turn. The factors which induce mesenchymal cells to modulate to osteoblasts and to deposit bone matrix in vivo have not been determined, except for those membrane bones surrounding the brain and forming the dermocranium and for the periosteal bones associated with the cartilage models of long bones. The intramembraneous (perichondral, periosteal bone) which forms around the shafts of endochondral bones has been

10 338 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. 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 surrounding the brain develop from mesenchyme whose skeletogenic potential 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- FIG. 6. The inductive interactions between brain, notochord and overlying mesenchyme responsible for formation of cranial dermal bones in embryonic chick. (From Schowing, 1968c.)

11 SKELETAL DIFFERENTIATION AND EVOLUTION 339 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 provide 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 H 3 -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,

12 340 BRIAN K. HALL similar stem cells form different skeletal tissues according to site-specific factors. Hall (1970, 1971, 1972) has reviewed many of the factors involved in such transformations and an example is discussed later. The sharks, despite their undoubted position as cartilaginous fishes, contain mesodermal cells capable of producing bone. Bone has been described in the modern sharks and chimeras (Peyer, 1968): at the base of the teeth in Squalus acanthias, where it is acellular (Moss, 1970), and in Heptanchus (Holmgren, 1942). Bone has also been observed in Ornithoprion hertwigi, a specialized Permian edestidaed shark. This species was partially armored, the armor consisting of denticles and scales, the bases of which were imbedded in bone. These bony elements were fused together to form a thick layer of bone around the snout and the mandible (Zangerl, 1966). Whether these bones are of neural crest origin is unknown. Whether they are or are not, they illustrate the hidden potential resident within skeletogenous cells, and indicate that views such as, "the process of bone development is, so to speak, much more firmly imbedded in the embryological pattern of these (Osteichthyes) fishes" (than in Chondrichthyes) (Romer, 1964, p. 76), are too restrictive in concept. Much of the skeleton of the early vertebrates was probably of neural crest origin, i.e., the homologous bones in present day vertebrates are derived from the cells of the neural crest. Romer (1972) has divided the skeleton into the predominantly cartilaginous "visceral skeleton" of neural crest origin and the predominately post-cranial "somatic skeleton" of mesodermal origin and has argued that the visceral skeleton antedated the somatic skeleton in vertebrate evolution. The neural crest must then have appeared very early in vertebrate evolution and its cells possessed the properties of migration, interaction with ectoderm, and modulation to different scleroblast types equally early. The development of epithelial-mesodermal interactions may have been a later development in vertebrate evolution. Maderson (1975) has reviewed the evidence for the homology of developmental processes in both fossil and recent vertebrates, and Jollie (1968) has provided an excellent discussion of the neural crest and of the consequences of acceptance of the Delamination Theory (to be discussed below). If the neural crest of the early vertebrates possessed the ability to form a variety of skeletal tissues, as appears, based on the tissues present in fossil Ordovician vertebrates, to be the case, then the skeleton of these vertebrates was a highly adaptive organ, ideally suited to evolutionary modification. Mute fossil bones do speak when asked the right questions. Delamination The previous studies provide experimental evidence for the delamination theory proposed by Holmgren (1940) and modified by Jarvik (1959) and Moss (1968c, 1969). In this theory it is postulated that dermal skeletal tissues, formed from the ectomesenchyme of the neural crest, lose their contact with the epidermis and sink in, to be replaced at the surface by a succeeding layer of skeletal tissues, not necessarily of the same type as the first-formed tissues. The first layers to sink in by delamination form the endoskeleton, the last-formed layers remain at the surface to become the dermal exoskeleton (Tarlo, 1964; Moss, 19686). Hay (1964) has shown that collagen, produced in the ectoderm, can migrate down into the underlying mesoderm and there contribute to the extracellular matrix produced by the scleroblasts a present day visualization of delamination. The exo- and endoskeletons and the various scleroblast types of the neural crest are thus linked by these common developmental processes. They may also be linked, as noted earlier, with inductive interactions with the underlying nervous tissue (Schowing, I968a,b,c). Ectodermal and mesodermal scleroblasts This discussion of the origin of skeletal tissues from the neural crest raises the question of the origin of skeletal tissues from the mesoderm and/or the ectoderm, for skeletal tissues are traditionally regarded as mesodermal tissues. As Mathews indicates (1967, p. 500): "Rigid adherence to re-

13 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, SKELETAL DIFFERENTIATION AND EVOLUTION a,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 AC- TIVITY 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

14 342 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. Moss (1969) has shown that, within the reptilian dermis, a wide range of skeletal tissues may be produced from similar presumptive cells. For example, all stages from tendon, through calcified tendon to bone, are found, and he has argued (1964a) that most primitive vertebrates also had the potential to produce the whole range of skeletal tissues during their evolutionselection pressure, and modulation dictati ng which type of tissue would actually form at a given site in a given species at a given time. The restriction in the range of skeletal tissues as one ascends the vertebrate phylogenetic tree indicates that particular skeletal tissues have become preferentially selected for during the course of evolution. However, even in those vertebrates which 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 significance of acellular bone remains unknown. The acellular bone of the teleosts develops in one of three ways from one of three types of cellular tissues: periosteal osteogenesis from the cells (osteoprogenitors) of the periosteum; tendonous osteogenesis from the cells within the tendons; or osteogenesis by metaplasia of secondary cartilage cells (Moss, 1964a). Thus, Moss (19646, p. 348) writes: "It is apparent that no neccessary relationship exists between the type of bone first formed, and the type which is eventually characteristic of the species." The osteocytes so-formed either are not enclosed in an osseous matrix or, if enclosed, are replaced by an osseous matrix, resulting in an acellular bony matrix in the adult. Similar transforma-

15 SKELETAL DIFFERENTIATION AND EVOLUTION 343 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 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 of such structures. Sesamoid bones are known to be intratendinous ossifications (Barnet and Lewis, 1958) or intraligamentous ossifications (Burton, 1973) and it is known that the ornithischian dinosaurs possessed ossified tendons (Haines, 1969) and that reptiles tend to show tendinous rather than periosteal ossification (Moss, 1969; Enlow, 1969). The ligaments and tendons arise from connective tissue along lines of stress (Schaeffer and Rosen, 1961) and local tensions apparently induce them to ossify (Haines and Mohuidiin, 1968). Thus, the formation of the sesamoids need not have involved the development of radically new processes of ossification, and they are not bones radically different in construction from other bones. Their evolution probably involved the adaptation of pre-existing processes in response to new environmental conditions (the extra stresses imposed on the joints by the adoption of terrestrial life). The formation of secondary centers of 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. SIZE AND SHAPE OF THE SKELETON We may now turn to the question: What determines the size and shape of the skeleton? Morphogenesis of the skeleton Once the cells of the skeleton begin to differentiate a three-dimensional structure develops. At first the cells within the condensed mesoderm are randomly arranged. As the cells begin to synthesize extracellular matrix and to differentiate, they elongate perpendicular to the Jong axis of the condensation, and so begin to initiate a pattern and direction to the growth and shape of the ruminent. From then on in the development of the skeletal rudiment factors other than the direct genetic constitution of the tissues begin to come into play. A study of the determination of the morphology of particular bones within the skeleton sheds considerable light on the relative contributions of genetic and

16 344 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 neccessary 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-

17 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. SKELETAL DIFFERENTIATION AND EVOLUTION 345 Single character analyses such as those of Bock and of Beecher (1950, 1951) and Cracraft (1968) serve to show the wide variation in morphology which may exist between closely related species and highlight that there are many ways by which skeletal elements may respond to environmental conditions. The final form of the character depends upon (at least): (i) selection acting directly on the character; (ii) influences from neighboring structures; (iii) chance factors (Cracraft, 1968). The establishment of contact of two previously 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? Phytogeny of skeletal tissues These studies illustrate the difficulties involved in establishing a phylogeny of skeletal tissues based on the genetic selection of progressively more well-adapted tissue types. A partial list of the epigenetic factors which can influence skeletal histogenesis and final structure and which presumably did so early in vertebrate evolution would include: rate of growth of

18 346 BRIAN K. HALL the bone, rate of growth of the rest of the functional unit, degree of remodeling of the bone, muscle attachments, degree of vascularization, mechanical factors, size of the animal, habitat (terrestrail, aquatic, aerial), length of the development period, age at maturity, seasonal feeding cycles, and hormonal milieu. When we say that no major structural advances were associated with the evolution of the skeleton and that a phylogeny of bone or of cartilage, involving progressive advancement with time cannot be established, we do not wish to imply that no changes occurred with time. Moss (1964a) has listed four trends in the evolution of the skeleton: decrease in the range of skeletal types; decrease in the amount of bone per animal; decrease in the number of bones per animal; and a more restricted location of bone (e.g., decrease in dermal armor). Note that none of these involve increased specializations of the skeletal tissues; if anything they reflect dimunition of skeletogenesis with time. Moss also lists three factors associated with evolution above the fish level which he considers as possibly responsible for the above changes: the skeleton becomes weight bearing, homeostatic for calcium and phosphorous, and a source of hematopoeitic cells. CONCLUDING COMMENTS The skeletal tissues of both fossil and recent vertebrates form a coherent, interrelated class, according to a number of criteria: embryological development, production of extracellular matrices consisting of a fibrous protein and a carbohydrate component, ability to calcify the matrix, the potential to modulate to other cell types, high degree of responsiveness to modification by environmental factors. There is, therefore, justification in speaking of skeletal tissues as closely related to one another and this has led to attempts to establish phylogenies of skeletal tissues. The data presented in this paper indicate that the very high degree of plasticity shown by these cells, their readiness to modulate in response to epigenetic factors, and their early appearance as specialized tissues in the vertebrate lineage, make such attempts difficult, and indeed, there may be no basis in fact for such evolutionary trends. The earliest vertebrates possessed highly specialized skeletal tissues and left evidence, in the form of tissues intermediate between those recognized as discrete endpoints (bone, cartilage, dentine) that their cells were highly adaptive even at the outset of vertebrate evolution. The readiness with which skeletal tissues respond to epigenetic factors provides considerable insight into the mechanisms whereby the skeleton and its associated soft tissues may have evolved. The skeletal tissues are supremely pre-adapted to exploit new environmental pressures. Convergence and divergence ought to be expected and according to Jollie (1968) occurred in the initial, separation of the gnathostomes from the agantha. The modulation of the scleroblasts to cartilage facilitated rapid embryonic growth. Modulation to bone facilitated storage of essential ions, bearing of increased weight, and the transition to land. These are some of the evolutionary consequences of skeletal differentiation. NOTE ADDED IN PROOF Recently, le Lievre (1974) has shown that both bone and cartilage of the avian visceral skeleton are derived from ectomesenchyme of the neural crest. REFERENCES Alexander, R. McN Evolution of integrated design. Amer. Zool. 15: Applegate, S. P A survey of shark hard parts. Pages in P. W. Gilbert, R. F. Mathewson, and D. P. Rail, eds., Sharks, skates and rays. Johns Hopkins Press, Baltimore. Barnett, C. H., and O. J. Lewis The evolution of some traction epiphyses in birds and mammals. J. Anat. 92: Beecher, W. J American Orioles. Wilson Bull. 62: Beecher, W. J Adaptation to food-getting in the American blackbirds. Auk 68: Beer, G. de Embryos and ancestors. 3rd ed. Oxford Univ. Press, London. Berrill, N.J The origin of the vertebrates. Oxford Univ. Press, London.

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