I. INTRODUCTION The purpose of this chapter is to describe the processing of pre-tRNAs in yeast, integrating information on processing in the nucleus and mitochondria. Precursor tRNAs produced from nuclear genes and mitochondrial genes have the same general requirements: Various activities are needed to remove 5´leaders and 3´trailers, to add the CCA end, and to catalyze numerous base modifications (Fig. 1). In addition, a subset of nuclear pre-tRNAs have introns that must be removed, although no such activities are required for the biogenesis of any yeast mitochondria1 tRNA. Some yeast nuclear tRNA genes are transcribed together in dimeric pairs (Schmidt et al. 1980), and mitochondria1 tRNAs are transcribed with other tRNAs (Palleschi et al. 1984b; Martin et al. 1985b; Bardonne et al. 1987; Francisci et al. 1987), with ribosomal RNAs (Osinga et al. 1984; Palleschi et al. 1984a), with mRNAs (Miller et al. 1983; Zassenhaus et al. 1984), or with the RNasc P RNA (Shu and Martin 1991). Although these polycistronic transcripts are processed by a variety of activities, only those directly involved in tRNA recognition and processing are considered here. We have not attempted to review tRNA gene organization or transcription, as these topics have been covered elsewhere. The reader is referred to Guthrie and Abelson (1982) for a review of yeast nuclear tRNA genes, to Thuriaux and Sentenac (this volume) for a review of nuclear tRNA gene transcription, and to Tzagoloff and Myers (1986) for a review of mitochondria1 tRNA genes. In general, we have...

The extracellular matrix (ECM) is a highly heterogeneous amalgam of multidomain molecules that are intimately involved in the development, growth, function, and homeostasis of every organ system, including the skeleton. Similar to other connective tissues, bone and cartilage matrices consist of collagens, proteoglycans (PGs), and noncollagenous (NC) proteins, in addition to including enzymes involved in matrix assembly and degradation. That the vast majority of these molecules are also found in other tissues indicates that relative differences in ECM composition specify form and function at discrete anatomical locations of the developing and adult skeleton. This chapter provides an introduction to ECM composition and organization in the skeleton, and a brief review of the contribution of selected matrix molecules to bone formation and remodeling that is mostly based on genetic evidence from loss-of-function studies in mice. Similar topics are also covered in other chapters of this book, and a number of excellent reviews are available that describe various aspects of ECM biology in greater detail. ECM COMPOSITION AND ORGANIZATION Collagens Collagens are the most abundant and diverse components of the connective tissue (Mecham 1998; Birk and Bruckner 2005). All collagens possess at least one triple helical (or collagenous [COL]) domain and NC domains of variable length and composition. Most collagens give rise to morphologically diverse suprastructures that are also referred to as molecular composites because they include additional collagens and NC proteins (Birk and Bruckner 2005). For example, tissue-specific organization of collagen I or II networks is largely regulated by copolymerization with...

The vertebrate skeleton is composed of approximately 200 bones, ranging in shape and size from the delicate bones of the mammalian inner ear to the robust femur. Each individual bone forms in a precise location and orientation with respect to its neighbors and in relation to force generating and transmitting tissues—the muscles, tendons, and ligaments. The appropriate structure of the bones is essential for function of the skeleton to support and move the body, and depends on an array of molecular cues that pattern their formation early in development. Our knowledge of developmental mechanism patterning all tissues and organs of the body, including the skeleton, is largely derived from experiments using two model systems—chick and mouse embryos. While aspects of patterning the craniofacial and axial skeletal elements have been elucidated, development of the bones of the limbs is particularly well understood. The limbs are easily accessible for embryological manipulation and are expendable for the survival of prenatal animals, allowing for analysis of late developmental phenotypes after genetic or surgical perturbation. The developing limb bud has therefore become an important model for the investigation of cellular and molecular mechanisms that pattern the tissues that give rise to bones. The tetrapod limb is of additional interest from an evolutionary perspective because it is a conserved but malleable structure whose adaptive variations in form increase an animal’s fitness in different ecological niches—by promoting mobility, aiding in the acquisition of food, fighting against or escaping from predators, and assisting in reproduction...

In contrast with chondrocyte differentiation, where all maturational stages are morphologically marked as well as spatially distinguishable within the growth plate, osteoblast differentiation is not marked by phenotypic changes in vivo, and osteoblasts in culture are, and remain throughout their differentiation, similar to fibroblasts. This absence of morphological features implies that one has to rely on gene expression studies to assess osteoblast differentiation. However, here again, the osteoblast has a poorly specific genetic program. Most of the proteins expressed by this cell type are also expressed in other cells, notably in fibroblasts. Another feature of osteoblast differentiation is that its embryonic layout is more complex than the events taking place once the skeleton is formed. Indeed, the developmental process by which osteoblast precursors first appear in the bone collar, begin to differentiate and then migrate within the core of the forming skeletal element along with invading blood vessels, is not observed anymore once the bones are formed. In the mature skeleton osteoblast, progenitor cells are spread out within the bone marrow and differentiate in situ. These two particularities explain for the most part why identifying the key transcriptional events required for osteoblast differentiation and function has been slower than for other cell types. However, in the last decade, these limitations have been overcome due to a combination of molecular efforts and genetic studies in mice and humans. This chapter summarizes our current knowledge about the transcriptional control of osteoblast differentiation and function (Fig. 1). CONTROL OF OSTEOBLAST DIFFERENTIATION BY RUNX2...

During the past 25 years, the use of genetic approaches has contributed substantially to the understanding of skeletal development and growth. Identification of mutations responsible for a large number of human osteochondrodysplasias and dysostoses (Mundlos and Olsen 1997a,b) has provided insights into the roles not only of individual genes, but also of entire developmental pathways. The correlation of clinical phenotypes with molecular alterations has allowed analyses of structure-function relationships. Coupled with studies of the phenotypic consequences of gene mutations in inbred mouse strains, and more recently also in zebrafish, such analyses have resulted in deep insights into the genetic mechanisms that underlie skeletal assembly, growth, maintenance, and functions. SKELETAL DEVELOPMENT AND GENETIC DISORDERS The vertebrate skeleton is the product of mesenchymal cells (osteochondroprogenitors of cartilage-forming chondrocytes and bone-forming osteoblasts) derived from cranial neural crest, paraxial mesoderm, and lateral plate mesoderm (Olsen et al. 2000). Bone marrow-derived myeloid cells are the progenitors for bone- and cartilage-resorbing cells, called osteoclasts. Neural crest cells give rise to the branchial arch derivatives of the craniofacial skeleton, paraxial mesoderm contributes to both the craniofacial and the axial skeleton, and the lateral plate mesoderm supplies progenitor cells for the limb skeleton (Fig. 1). Progenitor cells from these sources migrate into the regions in which future bones are formed, condense into elements of high cellular density, and differentiate into either osteoblasts or chondrocytes. Osteoblastic differentiation, followed by synthesis of bone extracellular matrix, occurs in regions of membranous ossification, such as the calvarium of the skull, the maxilla,...

Our understanding of skeletal biology has taken tremendous strides during the past few years. On the one hand, the spectacular recent breakthroughs in developmental biology have led to an understanding of the global rules shaping and positioning the cartilage and bone primordia in the vertebrate embryo. On the other hand, the discovery of key master regulators of the chondrocyte and bone lineage, such as Sox9 and Runx2, as well as the signaling pathways involved in the regulation of the differentiation of these lineages, has provided a much better understanding of these processes. This knowledge led to the elucidation of the molecular etiology of a majority of bone and cartilage genetic diseases. The goal of this book is to provide a comprehensive and up-to-date summary of the field of skeletal biology. This is a large field and due to space limitations, some areas might be covered more extensively than others. However, an attempt was made to cover all stages of skeletal development and patterning, as well as differentiation of cartilage and bone cells. The complex area of bone physiology is discussed in some of the chapters, but is not addressed extensively. This book covers essentially three major themes. The first theme relates to the development and patterning of bone in vertebrates. Several chapters deal with classical model systems that have been used to study bone and cartilage patterning in the vertebrate embryo. Specifically, the limb bud and the rules governing the formation of bone primordia are addressed, as well as the

Bone is a form of highly specialized mineralized connective tissue that provides strength to the skeletal system of higher vertebrates, while still retaining a certain degree of elasticity. The bone matrix is produced by osteoblasts, a cell-type that develops locally from mesenchymal precursors, and is resorbed by the osteoclast, a cell-type of hematopoietic origin. A few elements, such as the flat bones of the skull and part of the clavicle, are formed by the process of intramembranous ossification, whereby osteoblasts differentiate directly from cells within mesenchymal condensations. In contrast, the majority of skeletal elements are formed by endochondral ossification involving the remodeling of initial cartilaginous templates into bony tissue. The latter process requires controlled maturation of chondrocytes from proliferating and prehypertrophic to hypertrophic chondrocytes, as well as signaling from the prehypertrophic cells to the surrounding cells in the perichondrium, resulting in a regional induction of osteoblast differentiation. Osteoblasts start to differentiate in the periosteum, a region flanking prehypertrophic and hypertrophic chondrocytes. The typical appearance of one end of a juvenile long bone still containing a cartilaginous growth plate is shown in Figure 1. Recent lineage studies suggest that osteoblasts and chondrocytes share a common precursor in the limb. Thus, especially in the limb, the activation and/or inhibition of distinct signaling pathways is necessary in order to coordinate the differentiation of neighboring cells into distinct cell lineages and to synchronize their maturation. This chapter focuses on genetic and molecular studies elucidating the role of different locally produced growth factors during embryonic...

Osteoclast differentiation is an important biological process that determines the level of bone resorption in vivo. Numerous cytokines and growth factors are involved in the regulation of this process by directly acting on osteoclast precursor cells or through their effect on osteoclastogenesis-supporting mesenchymal cells, such as osteoblasts. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine that promotes osteoclastogenesis and, in most cases, the effects of other factors can be explained in the context of cross talk with RANKL signaling. This chapter describes recent advances in the understanding of the intracellular signaling mechanism of RANKL and its interaction with other signaling events during osteoclastogenesis, which may provide a molecular basis for therapeutic intervention in pathological bone resorption. THE ESSENTIAL ROLE OF THE RANKL-RANK SYSTEM IN OSTEOCLASTOGENESIS Osteoclast differentiation is a tightly regulated process because the balance between osteoclasts and osteoblasts is critical for bone homeostasis. Osteoclasts differentiate from hematopoietic cells of monocyte/macrophage lineage, but osteoclastogenesis-supporting cells of mesenchymal origin are required for the differentiation commitment. Thus, osteoclasts have traditionally been formed in a coculture of hematopoietic cells derived from bone marrow and calvarial osteoblasts, which were thought to express an unknown osteoclast differentiation factor. Before this factor was identified, information about the molecules involved in osteoclast differentiation had been obtained from the analysis of osteopetrotic mice (Fig. 1) (Asagiri and Takayanagi 2006). RANKL, a type II membrane protein of the TNF superfamily, was identified as the long sought-after osteoclast differentiation factor expressed by osteoblasts, but interestingly, the same molecule...

One of the most striking features of the human spine is its periodic organization. This so-called “segmental” arrangement of the vertebrae along the anteroposterior body axis is established during embryonic development. Structures called somites, which contain the precursors of the vertebrae, form in a rhythmic fashion at the posterior end of the embryo during the process of somitogenesis. Somites are sequentially added to the growing axis, thus establishing the characteristic periodic pattern of the future vertebral column. The primary segmentation of the vertebrate embryo displayed by somitic organization also underlies much of the segmental organization of the body, including muscles, nerves, and blood vessels. In amniotes, somites are the major component of the paraxial mesoderm that form bilaterally along the nerve cord as a result of primitive streak and tail bud regression during body axis formation. Somites bud off from the anterior presomitic mesoderm (PSM) as epithelial spheres surrounding a core of mesenchymal cells called the somitocoele. The dorsal portion of the somite remains epithelial and forms the dermomyotome, which differentiates into muscle and dermis while its ventral moiety undergoes an epithelio-mesenchymal transition, leading to the formation of the sclerotome. The sclerotome gives rise to the skeletal elements of the vertebral column: the vertebrae, ribs, intervertebral disks, and tendons. Most of our understanding of amniote somitogenesis at the morphogenetic and molecular levels results from studies involving the chicken ( Gallus gallus ) and the mouse ( Mus musculus ). In this chapter, we essentially focus on the patterning and development of the spine in...

HISTORICAL BACKGROUND One of the most striking characteristics about the craniofacial bones is that, contrary to the rest of the vertebrate skeleton, they are not entirely of mesodermal origin. Embryological studies, which started at the end of the 19th century with the observations of Kastschenko (1888, for selacians) and Goronovitch (1892, 1893, for teleosts and birds), have established that mesenchymal cells can arise, not only from the mesodermal, but also from the ectodermal germ layer. During this period, Julia Platt was the first to propose in 1893 that ectoderm contributed not only to the mesenchyme, but also to the cartilage of the visceral arches and to the dentine of the teeth in the mud puppy, Necturus . This derivation of mesenchyme, bones and cartilages from the ectoderm, was shown to occur via a transient structure, the Neural Crest (NC), which was first described in the chick embryo by the German Histologist Wilhem His in 1868. These observations contradicted the germ layer theory first put forward by Christian Heinrich Pander (1817), who described the formation of three layers of cells from the chick blastoderm. Later, Karl von Baer (1828) extended Pander’s findings to all vertebrate embryos. In 1849, Thomas Huxley generalized the presence of germ layers to invertebrates and the terms ectoderm, mesoderm , and endoderm were first used to designate the vertebrate germ layers by Ernst Haeckel in 1874, in the context of the Gastrea concept. The observation that formation of germ layers precedes organ morphogenesis and cellular differentiation was followed by...