Bones and Cartilage: Developmental and Evolutionary Skeletal Biology

Table of Contents

Cover image

Title page

Epigraph

Copyright

Preface

Abbreviations

Part I: Skeletal Tissues

Introduction to Skeletal Tissues

Chapter 1: Types of Skeletal Tissues

Chapter 2: Bone

Chapter 3: Cartilage

Part II: Natural Experiments

Introduction to Natural Experiments

Chapter 4: Invertebrate Cartilages

Chapter 5: Intermediate Tissues

Chapter 6: An Evolutionary Perspective

Part III: Unusual Modes of Skeletogenesis

Introduction to Unusual Modes of Skeletogenesis

Chapter 7: Horns and Ossicones

Chapter 8: Antlers

Chapter 9: Tendons and Sesamoids

Part IV: Stem Cells

Introduction to Stem Cells

Chapter 10: Embryonic Stem Cells

Chapter 11: Stem Cells in Adults

Part V: Skeletogenic Cells

Introduction to Skeletogenic Cells

Chapter 12: Osteo- and Chondroprogenitor Cells

Chapter 13: Dedifferentiation Provides Progenitor Cells for Jaws and Long Bones

Chapter 14: Dedifferentiation and Urodele Amphibian Limb Regeneration

Chapter 15: Cells to Make and Cells to Break

Part VI: Embryonic Origins

Introduction to Embryonic Origins

Chapter 16: Skeletal Origins: Somitic Mesoderm

Chapter 17: Skeletal Origins: Neural Crest

Chapter 18: Epithelial–Mesenchymal Interactions

Part VII: Getting Started

Introduction to Getting Started

Chapter 19: The Membranous Skeleton: Condensations

Chapter 20: From Condensation to Differentiation

Chapter 21: Skulls, Eyes and Ears: Condensations and Tissue Interactions

Part VIII: Similarity and Diversity

Introduction to Similarity and Diversity

Chapter 22: Chondrocyte Diversity

Chapter 23: Cartilage Diversity

Chapter 24: Osteoblast and Osteocyte Diversity

Chapter 25: Bone Diversity

Part IX: Maintaining Cartilage in Good Times and Bad

Introduction to Maintaining Cartilage in Good Times and Bad

Chapter 26: Maintaining Differentiated Chondrocytes

Chapter 27: Maintenance Awry – Achondroplasia

Chapter 28: Restarting Mammalian Articular Chondrocytes

Chapter 29: Repair of Fractures and Regeneration of Growth Plates

Part X: Growing Together

Introduction to Growing Together

Chapter 30: Initiating Skeletal Growth

Chapter 31: Form, Polarity and Long-Bone Growth

Chapter 32: Long Bone Growth: A Case of Crying Wolff?

Part XI: Staying Apart

Introduction to Staying Apart

Chapter 33: The Temporomandibular Joint and Synchondroses

Chapter 34: Sutures and Craniosynostosis

Part XII: Limb Buds

Introduction to Limb Buds

Chapter 35: The Limb Field and the AER

Chapter 36: Adding or Deleting an AER

Chapter 37: ERs in Limbed and Limbless Tetrapods

Part XIII: Limbs and Limb Skeletons

Introduction to Limbs and Limb Skeletons

Chapter 38: Axes and Polarity

Chapter 39: Patterning Limb Skeletons

Chapter 40: Before Limbs There Were Fins

Part XIV: Backbones and Tails

Introduction to Backbones and Tails

Chapter 41: Vertebral Chondrogenesis: Spontaneous or Not?

Chapter 42: The Search for the Magic Bullet

Chapter 43: Tail Buds, Tails and Taillessness

Part XV: Evolutionary Skeletal Biology

Introduction to Evolutionary Skeletal Biology

Chapter 44: Evolutionary Experimentation Revisited

References

Index

Epigraph

Problems of the developmental physiology of bone have always had a great fascination for those who have studied them. That fascination is likewise easily understood when we reflect how notable an example is provided by bone of the way in which, from the apparently simple, more complex problems arise, and how the pursuit of these further problems leads the investigator, if [s]he be so minded, straight to fundamental problems of the nature of life itself.

(Brash, 1934, p. 306)

The facts to which I wish to call your attention, and which are confirmatory of the views here developed, have been brought to light by different observers, at different times.

(Hubrecht, 1897, p. 37)

Copyright

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Preface

The skeleton has fascinated humankind ever since it was realized that, aside from one or several sets of genes, bare bones are our only bequest to posterity. But the skeleton is more than an articulated set of bones: its three-dimensional conformation establishes the basis of our physical appearance; its formation and rate of differentiation determine our shape and size at birth; its postnatal growth orders us among our contemporaries and sets our final stature; while its decline in later life is among the primary causes of loss of the swiftness and agility of youth. Not surprisingly, the skeleton is a central focus of many scientific and biomedical disciplines and investigations.

For the developmental or cell biologist, the skeleton provides an excellent model for studies of gene action, cell differentiation, morphogenesis, polarized growth, epithelial–mesenchymal interactions, programmed cell death, and the role of the extracellular matrix. The skeleton supplies the geneticist with a permanent record of the vicissitudes of its growth, whereby the phenotypic expression of genetic abnormalities can be studied. The orthopaedic surgeon earns a livelihood from correcting abnormalities and breaks, while the orthodontist corrects the position of teeth displaced consequent to alveolar bone dysfunction. Physiologists, biochemists and nutritionists are concerned with the skeleton’s store of calcium and phosphorus and its response to vitamins and hormones. Haematologists, on the other hand, find that the skeleton houses the progenitors of the blood cells. Pathologists endeavour to understand the disease states that result from abnormalities in skeletal cellular differentiation or function; surgeons want to prevent formation of skeletal tissues in the wounds that bear witness to their work. Vertebrate palaeontologists make their living from the analysis of the skeletons of extinct taxa. Veterinarians, physical anthropologists, radiographers, forensic scientists – the list goes on.

Bones come in all shapes and sizes. There are long bones, flat bones, curved bones, bones of irregular and geometrically indefinable shapes, large bones and small. Bones exhibit bumps, ridges, grooves, holes and depressions where they articulate with other bones, attach to tendons and ligaments, and where nerves and blood vessels course through them. Some bones and cartilages arise within the skeleton and are integral parts of it. Others arise outside the skeleton, some as sesamoids or ossifications within tendons or ligaments, others as pathological ossifications in what otherwise would be benign soft tissues. Bones and cartilages may develop during embryonic or foetal life, in larval stages or in adulthood – often late in adulthood – during normal ontogeny, wound repair, or regeneration. Bones modify themselves in response to injury, disease or parasitic infection, in the aftermath of surgery, as a defensive response to predators, as a consequence of domestication or hibernation, and through evolutionary adaptations.

My previous book on the skeleton – Developmental and Cellular Skeletal Biology – was published in 1978. That book concerned itself with how bones and cartilages are made and how these tissues, organs and systems evolved. So too does the present book, which includes and updates the earlier treatment. With respect to skeletal development, I address such questions as the following.

• Is bone always bone, no matter where and under what conditions it forms?

• Do bones that develop indirectly by replacing another tissue – be it cartilage, marrow, connective tissue, fat, tendon or ligament – differ from one another, and/or from bone that develops directly (intramembranously)?

• Is fast-growing the same as slow-growing bone?

• Is fish bone the same as human bone?

• Does bone form continuously or in cycles?

• Do bears make new bone during hibernation?

• Can sharks make bone?

• If cartilage does not contain type II (cartilage-type) collagen, is it still cartilage?

• Does the body contain cells that can differentiate as chondrocytes or as osteocytes and, if so, what factors allow cells to choose their fate?

• Are progenitor (stem) cells for bone and cartilage only found within the skeleton? If not, how do we recognize such cells and activate them for skeletogenesis?

• Why is aggregation (condensation) of cells so important for the initiation of the skeleton?

• Does the skeleton display daily or circadian rhythms?

• Do similar genes/growth factors regulate the differentiation of osteoblasts and chondroblasts?

• Can mononucleated cells resorb bone?

• How do joints form and remain patent?

• How does activating FGF receptors cause cranial sutures to fuse?

• What can mutants tell us about normal skeletogenesis?

• Does Wolff’s law really govern the structure of bone?

• How do chondroid, chondroid bone, osteoid and bone differ from one another?

• How do antlers, horns and knobs (ossicones) differ one from the other?

• Can we restart cell division in articular cartilage to effect repair?

With respect to the evolution of skeletal tissues, organs and systems, I ask such questions as the following.

• What are the evolutionary relationships between cartilage and bone and between acellular and cellular bone?

• How did novel features such as tetrapod limbs arise from fish fins?

• Can fossilized bone reveal patterns of growth, metabolism or physiology?

• Why are so few aware of the extensive cartilaginous skeletons found in many invertebrates?

• Is five the canonical number of tetrapod digits?

• If tetrapods are vertebrates with limbs, then how can limbless snakes be tetrapods?

• How did snakes lose their limbs?

• How did whales lose their hind limbs and transform their forelimbs into flippers?

• How do we recognize the diverse range of tissues in fossilized skeletons that are intermediate between connective tissues and cartilage, cartilage and bone, bone and dentine, or dentine and enamel?

• Why can some vertebrates regenerate their limbs or tails and others not?

• How does reduction in body size (miniaturization) affect the skeleton?

The answers to the above and many other questions may be found in this book. Sometimes the ‘answers’ are limited to descriptions. In other cases we have an extensive knowledge of the molecular, cellular, developmental and evolutionary processes involved. Some transitions (fins → limbs, for example) are understood in considerable detail, with paleontology, paleobiology, paleohistology, paleopathology, and the study of extant forms through molecular, cell and developmental biology contributing to our understanding. Other transitions – the origin of the turtle shell, for example – are much less well understood, with fossils contributing little and developmental information only beginning to appear.

Discussion of the mechanisms of skeletal development and evolution is organized into 15 parts to enable you to select with ease a topic of special interest. The range of skeletal tissues covered by the book is outlined in Part I. Although primarily devoted to bone and cartilage, Part I introduces dentine and enamel and four skeletal tissues that I call ‘intermediate’ because they display features of two or more of cartilage, bone, dentine and enamel. The four are chondroid, chondroid bone, cementum and enameloid. Discussion of these intermediate tissues is expanded in Part II in the context of what I refer to as ‘natural experiments,’ a category that includes invertebrate cartilages and an examination of the evolution of skeletal tissues.

Unusual tissues are followed in Part III by unusual modes of skeletogenesis, namely, horns, antlers, intratendinous ossifications and sesamoids, and the ossicones (knobs) of giraffes. Parts IV and V deal with the origin of skeletogenic cells, either as stem cells in embryos or adults (Chapters 10 and 11) or as more definitive skeletogenic cells (Part V). Here the emphasis is on those cells that can differentiate either as chondro- or osteoblasts (Chapter 12), on dedifferentiation as a source of skeletogenic cells in normally developing long bones and jaws and in regenerating urodele limbs (Chapters 13 and 14), and on the relationship(s) between the cells that make and the cells that break bone – osteoblasts and osteoclasts (Chapter 15).

I move explicitly into embryonic development in the three chapters in Part VI through examination of the embryonic origins of skeletogenic cells in somitic mesoderm and the neural crest, and an evaluation of the roles of epithelial–mesenchymal interactions in initiating skeletogenesis. The developmental processes that underpin skeletal formation – differentiation, morphogenesis and growth – are mediated through modification of cell division, movement, death (apoptosis) and/or specialization. To our amazement, similar genes and gene networks or pathways may be involved in all these developmental processes, and in vertebrates as diverse as sharks, salamanders, shrews and shore birds; pathways are conserved. In other instances, genetic pathways have diverged in different taxa; development evolves. Consequently, constancy, variation, change and novelty all confound our analyses. A major challenge for the future is to understand how conservation and/or modification at molecular, cellular and developmental levels produces and has produced the diversity of skeletal tissues, elements and systems seen in bone, cartilage and chondroid (tissues), the clavicle, humerus and ribs (elements), and in the endo- and exo-, appendicular, axial and craniofacial skeletal systems.

Skeletal development comprises a stepwise set of events, each depending on the step before, but each involving different cellular processes – migration, adhesion, proliferation, growth – and each subject to different genetic control. Condensation, the pivotal stage in the development of skeletal and other mesenchymal tissues, when a previously dispersed population of cells gathers together to differentiate into a single cell/tissue type such as cartilage, bone, muscle, tendon, kidney or lung, is the earliest stage during organ formation when tissue-specific genes are up-regulated. Condensation itself is a multistep process, involving initiation, establishment of boundary conditions, cell adhesion, proliferation, growth and cessation of growth. Chapters 19 and 20 in Part VII discuss condensation and the transition from condensation to overt differentiation of cartilage and bone, processes that are elaborated and related to epithelial–mesenchymal interactions in the context of a discussion of the development of the skull and optic and nasal capsules in Chapter 21.

Because cartilage is not cartilage is not cartilage, and an osteoblast is not an osteoblast is not an osteoblast, I devote Part VIII to an evaluation of similarity and diversity of cartilages and bones and of chondro- and osteogenic cells. Included in these chapters are discussions of the role of type X collagen, chondrocyte hypertrophy, vascularity and/or resistance to vascular invasion in different cartilages, and gender-based differences in the skeleton.

Part IX takes us to how chondrocytes are maintained as differentiated cells (Chapter 26), how chondrogenesis gone awry can lead to achondroplasia (Chapter 27), and to discussions of restarting chondrogenesis in articular cartilages or during bone repair and regeneration (Chapters 28 and 29).

Skeletal organs do not exist in isolation but are articulated at joints or sutures to form skeletal systems. How skeletal growth, especially long-bone growth, is initiated, how skeletal shape is maintained, and that perennial topic, Wolff’s law and the response of bone (and cartilage) to mechanical stimulation, are the topics of Part X; movement, maintenance of joints and abnormal fusion of sutures (craniosynostosis) are the topics of Part XI.

Although the search has been long, evolutionary developmental mechanisms are more elusive. Almost a century and a half ago, Thomas Henry Huxley recognized but three processes in the generation of animal morphology: (i) excess or (ii) suppression of one or more parts with respect to other parts, and (iii) the coalescence of parts. In essence, modification in a descendant of any of the developmental processes noted above and discussed in the text could lead to evolutionary changes in the skeleton. Reduce the size of a limb bud and digit number falls below five. Expand the width of the limb bud and an extra digit (polydactyly) can result. Limblessness and taillessness often result, not from failure to initiate a limb or tail bud, but from the inability to maintain the bud for long enough for skeletal tissues/organs to form. A mutation that allows limb buds to persist in an individual limbless tetrapod can result in the formation of an atavistic skeletal element. Add natural selection and a limbless taxon can result.

The six chapters in Parts XII and XIII discuss is some details limb buds, the limb skeleton, limbless tetrapods, fins, and the transformation of fins into limbs with the evolutionary origin of the tetrapods. Many are familiar with the apical ectoderm ridge or AER. Fewer will know that developing tails possess a ventral ectodermal ridge or VER. Part XIV is taken up with development of the vertebrae, including those of the tail, and including a discussion of tailless vertebrates in which the VER fails to function normally. Part XV – the last – explicitly treats evolutionary skeletal biology with discussions of skeletal variation, heterochrony, miniaturization, the evolution of novel skeletal elements (neomorphs) and atavisms.

Skeletal biology is a vast field; the research of thousands of individuals is included in the close to 6800 references cited. Nonetheless, and although I have endeavoured to be comprehensive, I have not been inclusive. I would appreciate important references I have omitted being brought to my attention.

Important conclusions are highlighted in the text. Topics that apply throughout the book and to which you the reader may want to refer frequently – the major proteins of bone, cartilage mineralization, Bmps and their receptors, as three examples – along with unexpected findings (chondrocytes with cilia, cartilage inside the notochord, the evolutionary consequences of hunting big sheep, cartilages and bones in the heart, how hibernation affects the skeleton, coal trimmers and shoemakers, fish without tails) are placed in boxes, which can be read as part of the text or in isolation. To make the text as user friendly as possible I have placed references, comments, elaborations, asides and tit(tid)bitsain endnotes, gathered together at the end of the text.

A device I have used to show how our understanding of a topic has evolved and how research programmes develop is to outline the research from a research group in a single list in chronological sequence. Furthermore, individual studies often pertain with equal utility to several aspects of, or approaches to, skeletal biology. Because of this, and because I want each section to be as self-contained as possible, you will occasionally find that I have repeated an example, explanation or mechanism. This does not reflect sloppy copy-editing. Rather, it is a deliberate way of demonstrating how interrelated the apparently separate topics are.

Although I want you to read the book, you should be able to access information readily through the index. Consequently, I have provided a detailed index, which serves as both subject and taxonomic index. The annotated list of abbreviations provided also can be used as a glossary of the many genes and growth factors discussed.

Inclusion of a list of abbreviations raises the issue of gene nomenclature.

Because of the rampant confusion and variation in the literature, a comment on the notation used for genes and their products has become a standard element in the preface of many recent books – Weiss and Buchanan (2004) for example, or been extended to an extensive appendix – Appendix 1 in Wilkins (2002) for example. Gene, growth factor and receptor nomenclature are in considerable disarray – Older names have been supplanted by newer names. And, that’s a good thing; it reflects the recognition of families of growth factors and genes and a consequent rationalization of nomenclature. So GHox-4.6 and GHox-8 isolated from chick embryos are now known to be a member of the Hoxd group (Hoxd-11) and an Msx gene (Msx-2), respectively. Independent and often contemporaneous discovery of the same gene or gene product in different laboratories introduced into the literature different names for the same entity. Examples are osteogenin, Bmp2B and rhBmp2B (human recombinant Bmp2B) for what is now known as Bmp-3. Even so, one has to be on one’s guard; older names remain in use in recent literature. I have endeavoured to provide current nomenclature throughout, including older names where appropriate.b Genes are in italics (Fgf-4), gene products in plain typeface (Fgf-4). Following convention, human genes or their products are capitalized (FGF-4, FGF-4).

My own interest in the skeleton was kindled by my Ph.D. supervisor, the late P. D. F. Murray, whose 1936 monograph Bones, A Study of the Development and Structure of the Vertebrate Skeleton remains one of the most lucid and, paradoxically, most modern treatments of the developing skeleton. The present book was meant to be a post-retirement project, one that I could dawdle over into my declining years. Charles (Chuck) Crumly, then of Academic Press, convinced me that I should make an earlier start on the project by providing a contract with a long lead-time and then believed me when I said I was working on the book and not dawdling … My thanks Chuck, for your faith, encouragement, and friendship. Completion of the book was aided enormously by the granting of a Killam Research Fellowship by the Canada Council for the Arts in 2003. Without the consequent release-time from teaching and administration I would be even further behind the generous deadline given when I signed the contract.

I am grateful that Tim Fedak applied his considerable artistic skills to the preparation of the figures, which enhance the text enormously. My thanks to Hollie Knoll, who prepared the majority of the tables (often from quite raw starting material), and to Patricia (Paty) Avendaño for assistance with some of the tables and for providing Figures 40.6 to 40.8. A number of other friends and colleagues kindly made figures available. My thanks to Michael Locke for Figure 2.3, Eckhard Witten for Figure 2.11, Tamara Franz-Odendaal and Andrew Gillis for the figures of scleral ossicles in salmon (Fig. 21.1), David Precious for Figure 33.2, and Ulrich Zeller for Figure 44.1.

Although I hope it is not too obvious from the book, it is not possible for one person to be expert in all the areas of skeletal biology. Skeletal biology is a vast field; the research of thousands of individuals is included in the close to 6800 reference cited. I am grateful to a number of experts in specific areas who took the time and trouble to respond to my request to review individual chapters. With the chapter they reviewed in parentheses, my thanks to Mike Benjamin (9), George Bernard (2), John Bertram (32), Bobo Christ (16), Nelly Farnum (31), Benedikt Hallgrímsson (44), Greg Handrigan (43), Tuomo Kantomaa (33), Gillian Morriss-Kay (34), Lynne Opperman (34), Pertti Pirttiniemi (33), Robin Poole (3), Cheryll Tickle (38) and Eckhard Witten (1–6). Tim Fedak, Tamara Franz-Odendaal and Matt Vickaryous, three current members of my laboratory, each provided comments on several chapters and engaged in a scavenger hunt for elusive titbits. Allison Cole commented on Chapter 4. Annie Burke, Andy Horn and Marty Leonard, Peggy Kirby, David Precious and Marvalee Wake provided information concerning somite organization, the identification of the species in Figure 7.7, cells migrating from the ventral neural tube, nasal septal growth, and chondroid in caecilians, respectively. Tom MacRae and Vett Lloyd kindly checked the list of abbreviations. It is a pleasure to thank Priscilla Goldby for expert copy-editing and Pauline Sones and Andy Richford of Elsevier for their professionalism in bringing the book into production. June Hall read two drafts of the entire book – each of which I thought was the penultimate draft – and provided much excellent advice, more of which, as always, I should have taken.

Since 1968 my research has been supported continuously by the National Research Council (NRC) and then the Natural Sciences and Engineering Research Council (NSERC) of Canada (grants A5056 and 257447-02), with additional support from time to time from the Research Development Fund and Killam Trust of Dalhousie University and from funds associated with the George S. Campbell Chair in Biology and a University Research Professorship, the Victoria General Hospital (Halifax), the Medical Research Council (Canada), the Canadian Institutes of Health Research, the Killam Trust of the Canada Council for the Arts, and the US National Institutes of Health (NIH; grant 45344). To all these agencies, my heartfelt thanks.

a Tidbit, US variant of titbit. Titbit, a dainty morsel, a piquant item of news. ‘The book is chock-full of colorful titbits about theater and theater people’ (Alec Guinness) is an example of the use of the term found on many web sites.

b M. P. Smith (1992) and S. Stein et al. (1996) provide guides and checklists to the vertebrate homeobox (Hox) genes, Duboule (1994) a guide to those homeobox genes known a decade ago. The rationale for gene nomenclature outlined by Wilkins (2000, pp. 525–526) is eminently sensible and rational; Wendy Olson and I followed this convention for Keywords & Concepts in Evolutionary Developmental Biology (Hall and Olson, 2003, p. xvi) and I use it here.

Abbreviations

Because acronyms and abbreviations abound, especially in molecular biology, metabolic pathways and enzymology, I provide a list of the abbreviations used in the book, including abbreviations used in tables but excluding those used in figures; many of the latter pertain only to a single figure legend. Where I felt it would be helpful I have annotated the explanation of an abbreviation. Entries marked * (e.g. *Fgf) represent families of molecules. In such entries, individual family members are not listed, but their identity in the text should be obvious. For example Fgf-1 and Fgf-2 – fibroblast growth factor one and fibroblast growth factor two – are two members of the Fgf family, BmpR-1B is a BmpR (bone morphogenetic protein receptor) and so forth. When only a single member of a family is mentioned in the book I only list that member. Bapx-1 and Gli-3 are two examples.

Genes and gene products are listed under the same abbreviation with the gene name in italics and the gene product in plain text as per convention, e.g. Fgf, Fgf. Gene names in humans are capitalized (FGF-2) in the text, but not listed separately in these abbreviations. Gene names for Hox genes were regularized a little over a decade ago. Nevertheless, and for ease of reference, I have included older names in this list and cross-listed them to the newer name, e.g. Hox 1.1 = Hoxa-7, Quox 7 = Msx-2.

Part I

Skeletal Tissues

Introduction to Skeletal Tissues

‘When Tess made me too weepy, I turned to the timeless serenity of the frontal bones’

(Bellairs, 1989, p. 93).

Acellular bone links evolutionary and developmental studies on the one hand, and normality and pathology on the other.

Types of Skeletal Tissues

‘In the pioneering stages of Natural Science we recognize the work of collecting, describing and classifying the typical units as a fundamental necessity. The study of their morphology and their history belongs to a more advanced period.’¹

Skeletal tissues are ancient, their origins reaching back perhaps three-quarters of a billion years. The number of skeletal tissues or organs is far more limited. In 2000, Thomas and colleagues evaluated 182 characters of skeletal design as possible design options in morphospace, a 3D representation of the distribution of all known morphologies. Of these 182 characters, 146 were already in use in animals of the Burgess Shale fauna 530 mya. Indeed, within 15 million years of the appearance of the crown groups of the major phyla, 80 per cent of the design elements were already in use.²

Four classes of mineralized tissues are found in vertebrates. The four are bone, cartilage, dentine and enamel. This may seem an unlikely list. Normally, we think of cartilage and bone as skeletal tissues, enamel and dentine as dental tissues. But enamel and dentine arose evolutionarily as skeletal tissues in the exoskeleton (dermal, dermoskeleton) of early vertebrates (Fig. 1.1, Table 1.1). Bone is also a primitive tissue of that exoskeleton. Cartilage, on the other hand, provided the basis for the second vertebrate skeletal system, the endoskeleton (Table 1.1), and has an even more diverse distribution and, potentially, a longer evolutionary history than bone.³ This is because cartilage and cartilage-like tissues form endoskeletal elements in many invertebrates. Although most invertebrates have non-cartilaginous endoskeletons the diversity of taxa with cartilage is astonishing. Where there is an exoskeleton or cuticle in invertebrates – and there almost always is – it is composed of chitin (sometimes with glycoproteins, sometimes mineralized) or calcium carbonate, but not calcium phosphate, which is the major component of bone.⁴ Neither bone nor mineralized cartilage is ever found in invertebrates, although some invertebrate cartilages have surprisingly ‘bone-like’ features (Chapter 4).

Figure 1.1 Diagrammatic representations of the tissues that comprise (A) the dermal (exo) and (B) the endoskeletons of vertebrates. The major difference is that the dermal skeleton is based on dentine and associated bone, while the endoskeleton is based on cartilage. (A) Enamel caps the dentine in the dermal skeleton. Individual exoskeletal units (odontodes), which produce dentine and bone of attachment, fuse to adjacent basal bone. (B) The cartilage of the endoskeleton may (i) remain unmineralized, (ii) mineralize and remain as a permanent mineralized (calcified) cartilage, (iii) be surrounded by perichondrial bone, or (iv) be invaded and replaced by endochondral bone.

Table 1.1 Definitions of terms for skeletal systems and modes of ossificationa

a Based on Hilton and William (1999) to which I have added modes of ossification. Although these terms were outlined in the context of a comparative analysis of fish skeletons and skeletal development they apply across the vertebrates.

Mineralization is not the exclusive property of the four vertebrate mineralized tissues. Mineralization is ubiquitous within metazoans as well as being a property of many single-celled organisms. In Table 1.2, I summarize the range and diversity of mineralized biological tissues in various groups and the major organic component(s) associated with mineralization in each. Mineralization can place enormous demands on an organism, for example when deer regrow their annual set of antlers (Chapter 8) or hens lay eggs (Chapter 25).

Table 1.2 The diversity of mineralization of biological tissuesa

These four broad classes of vertebrate skeletal tissues may be further subdivided in ways that reflect the interests of individual skeletal biologists and the scope of their fields. Embryologists subdivide skeletal tissues by developmental process, anatomists by structure, pathologists on the basis of deviation from the norm, and so on.

For vertebrate skeletons, I add a fifth ‘catch-all’ category to encompass those tissues that, on the basis of one or more criteria, are intermediate between two of the four mineralized tissues.⁵ Examples are tissues intermediate between bone and cartilage (chondroid, chondroid bone), bone and dentine (osteodentine, cementum), enamel and dentine (enameloid). The usefulness of this category for understanding the dynamic development of skeletal tissues will become apparent as we proceed. But first, I introduce the players.

BONE

Bone is a vascularized, supporting skeletal tissue – although it may also arise ectopically⁶ outside the skeleton – consisting of cells and a mineralized extracellular matrix (ECM). Bone is deposited by bone-forming cells (osteoblasts) and by osteocytes, some of which are ciliated (Box 1.1). Osteoblasts cease dividing when they transform into osteocytes. Bone is modeled, remodeled and/or removed by mono- or multinucleated osteoclasts (and sometimes by osteocytes). Type I collagen, composed of two αI chains and one αII chain (products of the Col1a1 and Col2a1 genes, respectively) and depicted as α1 (II)3, is the major extracellular matrix component. Osteocalcin (bone gla protein), osteopontin and osteonectin are the major non-collagenous proteins (Box 1.2). Bone matrix is permeated by canals (canaliculi) which contain osteocyte processes, and which connect to other osteocytes, to osteoblasts and to osteogenic cells on the surface via gap junctions, forming a syncytium. The fluid content of bone is low. Embryologically, bone arises from mesoderm and from neural crest, two of the four germ layers of vertebrate embryos.

Box 1.1 Cilia and skeletal cells

Chondroblasts/cytes and osteoblasts/cytes are embedded within matrices of varying degrees of fluidity, viscosity or solidity. Cilia are organelles that project from cells or single-celled organisms. Projecting into the external environment or into a lumen such as the gut, cilia function to move currents or particles (including food) or to move the entire organism. The notion of chondrogenic or osteogenic cells possessing cilia is the histologist’s version of an oxymoron. Nevertheless, cilia have been described on cartilage and bone cells.

Cells that clearly are osteocytes in rat and chick calvariae possess cilia, usually one per cell, one speculation being that cilia function to move fluids through the canaliculi (Fig. 1.2). Given that osteocytes reside within lacunae (Figs 1.3 and 1.4) this is not an implausible function.

Cells that clearly are chondrocytes in mouse radii, and rat and canine articular chondrocytes, also each possesses a single cilium (Fig. 1.5).a

a See Federman and Nichols (1974) and Tenenbaum et al. (1986) for cilia on osteocytes, and Scherft and Daems (1967), Wilsman and Fletcher (1978) and Vidinov and Vasilev (1985) for cilia on chondrocytes. See the chapters in Volume 1: The Osteoblast and Osteoclast of Hall (1990–1994) for overviews of the basic structure and function of osteoblasts and osteocytes.

Figure 1.2 Cilia and osteocytes. Occasionally, osteocytes display cilia. (A) A transmission electron micrograph of a cilium projecting from a rat osteocyte. (B) A cross section of the basal body shows the typical 9 + 0 fibril arrangement of cilia

Modified from Federman and Nichols (1974).

Figure 1.3 Osteocyte formation, as seen on the endosteal surface of a rat femur visualized with scanning electron microscopy. (A) Surface osteoblasts in the earliest stage of lacuna formation. Note the extensive and branched cell processes (arrows). (B, C) Further development of the lacuna with collagen fibrils in the lacunar wall and cell processes entering the pores (future canaliculi; arrows in B) in the wall of the lacuna. (D) An osteoblast depositing and being incorporated into osteoid.

Modified from Menton et al. (1984).

Figure 1.4 Scanning electron micrographs of osteocytes from the dentary of a 70-year-old human. These methacrylate replicas show the lacunae in which the osteocytes resided and the canaliculi through which osteocyte cell processes ran. The osteocyte in the inset on the left is enlarged on the right and shown on the cover.

Modified from Atkinson and Hallsworth (1983).

Box 1.2 Major non-collagenous proteins of bone matrix

Osteocalcin

Osteocalcin (bone γ-carboxyglutamic acid, Gla; bone Gla protein, BGP) is a 5800 MW, vitamin K-dependent, γ-carboxyglutamic acid-containing, Ca++-binding protein. The seventh most abundant protein in human bone, osteocalcin is the most abundant non-collagenous protein in bone, accounting for 10–20 per cent of the non-collagenous protein. Levels in human serum are 7.0 ± 2.5 ng/ml. Osteocalcin recruits osteoclasts or osteoclast precursors to bone for resorption. See Box 24.1 for further details.

Osteopontin

Osteopontin, a 66-kDa glycosylated phosphoprotein synthesized by osteoblasts, osteoclasts and macrophages, is found at active sites of bone metabolism: endochondral and membrane bone, osteoid, preosteoblasts, osteoblasts and osteocytes (Fig. 1.6). Osteopontin enhances cell survival and migration but inhibits mineralization. Positive and negative regulation of transcription, and regulation of osteocalcin and osteopontin protein levels have been described.a See Box 24.2 for further details.

Figure 1.6 Patterns of expression of osteopontin and osteonectin during murine limb development. (A) A longitudinal section of the forelimb from a 15.5-day-old embryo showing osteopontin (black) within the epiphyseal cartilage (arrowhead). (B) A section near that of A showing osteonectin transcripts (black) in the periosteum, subperiosteal bone, and in a developing tendon (arrowhead). (C) A higher magnification of part of A showing osteopontin transcripts (black), where endochondral ossification has been initiated, and absence of expression in the other chondrocyte zones. (D) A higher magnification of part of B showing osteonectin transcripts (black) in the periosteum (arrowheads).

Modified from Nomura et al. (1988).

Osteonectin

Osteonectin (or SPARC; secreted protein, acidic, cysteine-rich), a 32 000 MW extracellular matrix protein comprises some ten per cent of the protein in bone. Appearing with mineralization (Fig. 1.6), osteonectin links collagen to hydroxyapatite, serves as a nucleus for mineralization, and regulates the formation and growth of hydroxyapatite crystals. See Box 24.3 for further details.

a See T. A. Owens et al. (1991), Ayad et al. (1994) and Yagami et al. (1999) for osteocalcin and osteopontin.

The first bone matrix deposited is unmineralized and known as osteoid. Subsequently, osteoid is impregnated with hydroxyapatite to form bone, the mineralized tissue. An organic layer – the lamina limitans – separates osteoid from already mineralized bone (Scherft, 1978).

Bone is an aerobic tissue with high oxygen consumption. Bone functions to support the body, protect major organs such as the brain and spinal cord, and as a site of attachment for ligaments and muscles. Bone is also a storehouse for calcium and phosphorus, and a major site for the metabolic regulation of mineral homeostasis. Bone houses the haematopoietic tissues of adult mammals.

Bone is found only in vertebrates. Extant jawless vertebrates (lampreys), craniates (hagfishes) and all invertebrates lack bone. Conodonts possess bone; their phylogenetic status is not resolved to everyone’s satisfaction, however.

CARTILAGE

Cartilage is an avascular, supporting and articular skeletal tissue consisting of cells in an extracellular matrix (ECM), which may or may not mineralize depending on cartilage type. Like bone, cartilage can arise ectopically outside the skeleton, for example, in connective tissue, muscle and the heart. Cartilage is deposited by cartilage-forming cells (chondroblasts, chondrocytes; Fig. 1.7) and removed by mono- and multinucleated chondroclasts. Cartilage cells are separated from one another by pericellular and extracellular matrices. Unlike bone cells, chondrocytes lack connecting cell processes. Some chondrocytes are ciliated (see Box 1.1, Fig. 1.5). Most chondrocytes continue to divide throughout life, although in some cartilages (mammalian articular cartilages, for example), the number of dividing cells may be less than one per cent of the chondrocyte population.

Figure 1.7 A scanning electron micrograph of a chondrocyte in the articular cartilage of a human patella. The lacuna and cell processes are shown especially well

From Hall (1978a).

Figure 1.5 Cilia in cartilage. A transmission electron micrograph of an articular chondrocyte from the humerus of a two-day-old Labrador pup, with cilium and basal body attached to a centriole (A) and sets of microtubule triplets in the basal body (B).

Modified from Wilsman and Fletcher (1978).

The hydrated ECM of vertebrate cartilage is primarily composed of glycosaminoglycans (GAGs), notably chondroitin sulphates and proteoglycans. The major collagen is type II, composed of three αII chains, depicted as α1(I)2αII. Some types of vertebrate cartilages contain additional collagens; for example, type I in articular, fibro-and secondary cartilages, and type X in hypertrophic cartilage (Fig. 1.8).

Figure 1.8 Histological sections of articular cartilages from rat tibiae at one (A, D), five (B, E) and 11 (C, F) weeks after birth to show the distribution of type II collagen (A–C) and of type I collagen (D–F) as visualized with antibodies. Type II collagen is distributed throughout the cartilage matrix (A–C). Type I collagen is preferentially expressed in the superficial chondrocytes at the articular surface (D–F, arrows).

Adapted from Sasano et al. (1996).

Lamprey and hagfish cartilages lack collagen type II and have different classes of fibrous proteins (lamprins, myxin) in place of collagen. Cartilages of living agnathans do not mineralize in vivo. Within invertebrates, cartilaginous extracellular matrix is composed of GAGs and a modified form of type I collagen. No invertebrate cartilages mineralize. Vertebrate cartilages can arise from mesodermal and from neural crest-derived mesenchyme. The origins of invertebrate cartilages are less well known, although some may be epithelial.

Cartilage resists pressure, has a high fluid content, is anaerobic as a tissue, and so has low oxygen consumption. Cartilage functions as the embryonic endoskeletal tissue in vertebrate embryos (primary cartilage) and as an endoskeleton in many invertebrates. In vertebrates, remnants of primary cartilage transform to articular cartilage and function as the articular tissue at joints in endochondral bones (Figs 1.8 and 1.9; see Fig. 3.11), while secondary cartilage forms on many membrane bones in birds and mammals.

Figure 1.9 Development of the mandibular joint in the Japanese medaka, Oryzias latipes. Two stages post-fertilization (pf) and an adult are shown as longitudinal histological sections. Meckel’s cartilage is the element on the right in each. (A) six days pf; (B) 49 days pf; (C) adult. The position of the joint cavity is evident at six days pf (A) and is well developed by 49 days (B).

Photographed by Tom Miyake.

DENTINE

Dentine, a tubular, mineralized, dental and skeletal tissue, comprises the bulk of true teeth, true teeth being those composed of enamel and dentine, in contrast to keratinized structures that function as ‘teeth’ in, for example, anuran amphibian tadpoles. Dentine is the primary tissue of the vertebrate exoskeleton, and