Chapter Twelve - Mechanistic insights into skeletal development gained from genetic disorders

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Abstract

A complex cascade of highly regulated processes of cell fate determination, differentiation, proliferation and transdifferentiation dictate the patterning, morphogenesis and growth of the vertebrate skeleton, perturbation of which results in malformation. In humans over 450 different dysplasias involving the skeletal system constitute a significant fraction of documented Mendelian disorders. The combination of clinical, phenotypic characterization of rare human skeletal dysmorphologies, the discovery of causative mutations and functional validation in animal models has contributed enormously to the understanding of molecular control of skeletal development. These studies revealed a myriad of genes and pathways, such as WNT, Hedgehog (HH), planar cell polarity and primary cilia, as key regulators for skeletal patterning, growth and homeostasis. The generation of mouse models recapitulating human congenital skeletal dysplasia has provided mechanistic insights into the diverse pathologies caused by single gene mutations, integrated action of developmental pathways such as WNT and HH and the role of stress responses. Technological developments in whole genome and exome sequencing have accelerated the discovery of disease-causing mutations and are changing approaches for diagnosis. The discovery that non-coding variants and disorganization of the 3D genome are associated with limb patterning disorders has revealed an additional level of complexity in the regulatory framework of skeletal development and disease mechanisms. This chapter focuses on a selection of human skeletal pathologies which illustrate how new findings about the coding and noncoding genome, combined with functional modeling, are contributing to deeper understanding of skeletal development, mechanisms of disease, with therapeutic potential for chondrodysplasias.

Introduction

Congenital skeletal dysplasia arises when genetic alterations result in the disruption of the pattern, structure and growth of the skeleton. Such perturbations manifest as one or more phenotypes affecting the shape and size of individual skeletal elements, such as short, stubby fingers, duplications of fingers or toes, clubfeet, missing bones, fragile bones or curved spines. Normal skeletogenesis requires spatial and temporal control and integration of various transcription factors and signaling pathways to coordinate precisely the initial condensation of mesenchymal cells, specification of osteo-chondroprogenitors and the sequential phases of chondrocyte differentiation, proliferation, cell cycle exit and maturation, hypertrophy and the transition to the osteoblast lineage. Several of these factors are described in detail in other chapters. Many genes and pathways were discovered through identifying causative mutations associated with human skeletal syndromes. Advances and affordable technologies for sequencing whole genomes have accelerated the discovery of new genes and genetic loci in skeletal dysplasias (Bonafe et al., 2015; Geister & Camper, 2015). In this chapter, we highlight the contribution of recent discoveries of causative mutations in human skeletal dysplasias, combined with functional genomics, to the identification of key genes and pathways and gene regulatory mechanisms that govern different phases of skeletal development (Fig. 1). We also briefly illustrate how knowledge of the underlying molecular pathogenesis is being exploited for clinical translation, leading to human trials on chondrodysplasias.

Section snippets

Genetic control of patterning the appendicular skeleton

Human limbs consist of bones and soft tissues of particular size and shape arranged in a precise pattern. Structural abnormalities are often unique and diagnostic. One of the most recognizable limb phenotypes is polydactyly (Greek for “many fingers”). Digits in human hands and feet are formed in a highly conserved pentadactyl pattern, but individuals with polydactyly have additional digits arising on the side of the thumb (preaxial), the little finger (postaxial), or the central fingers

Skeletal morphogenesis: Integrated control of chondrocyte differentiation

The cascade of differentiation steps in endochondral bone development is controlled by a combination of key transcription factors and the integrated action of signaling pathways, as exemplified by a number of skeletal dysplasias. The discovery of SOX9 as a master regulator of chondrogenesis came from the identification of its causative role in Campomelic dysplasia (CD, OMIM#114290), a rare, semi-lethal autosomal dominant congenital skeletal disorder that affects approximately 1 in 40,000 to

Integrated signaling control of osteoblast differentiation and activity

The mechanisms underlying several bone disorders highlight the integration of signaling pathways in controlling bone formation. Progressive osseous heteroplasia (POH, OMIM#166350) is an autosomal dominant disorder characterized by widespread and disabling heterotopic ossification of skeletal muscle and deep connective tissues. It is caused by a null mutation of GNAS, which encodes Gαs, a protein that transduces signals from G protein-coupled receptors (Shore et al., 2002). In contrast,

Ciliopathies and the primary cilia in skeletal development

In the past decade, primary cilia emerged as important modulators of vertebrate HH signaling and their dysfunction has been linked to a spectrum of human diseases, collectively termed ciliopathies (Huber & Cormier-Daire, 2012). As cilia are a component of almost all cells, ciliary dysfunction often affects multiple organs and the phenotypic outcome is characteristic of aberrant HH signaling (Waters & Beales, 2011). For example, Meckel's syndrome (MES, OMIM#249000), Bardet-Biedl syndrome (BBS,

Planar cell polarity in the development of growth plate

The Planar Cell Polarity (PCP) pathway controls the process of convergent extension and collective cell migration and thereby the elongation of the body axis and shapes of many organs (Henderson, Long, & Dean, 2018). In endochondral ossification, the growth plate architecture of organized columns of cells requires PCP activity and its defects predispose humans to various skeletal dysplasias (Wang, Sinha, Jiao, Serra, & Wang, 2011). Robinow syndrome (RS) is a genetically and phenotypically

The impact of ER stress signaling on chondrocyte differentiation

The different cell types in the mammalian skeleton are embedded in tissue-characteristic complex extracellular matrix (ECM) networks, composed of collagens, proteoglycans, glycosaminoglycans, and glycoproteins. The ECM is important not only in providing structural support but by influencing cell adhesion, proliferation, migration, survival, differentiation and control of cell fate and morphogenesis. The pivotal role of the ECM is reflected in the major contribution of disruption in genes

Non-coding mutations and regulatory control of skeletal development

The discovery of mutations in non-coding genomic regions that cause skeletal dysplasia has brought a new dimension to our understanding of the regulatory control of skeletogenesis (Fig. 2). An outstanding example is the identification of a highly conserved cis-regulatory element within the preaxial polydactyly (PPD) transcription-associated region, called the ZPA regulatory sequence (ZRS). This enhancer is responsible for the initiation and spatially restricted expression of Shh in the ZPA,

Impacting 3D genome folding in skeletal disorders

Genomic DNA in the nucleus is organized as topologically associating domains (TADs), which are fundamental structural units that guide the physical interaction between cis-regulatory elements and promoters while insulating adjacent domains from inappropriate contacts (Dixon et al., 2012; Rao et al., 2014). Structural variations caused by chromosomal rearrangements or insertions or deletions affecting TADs can have profound effects on gene regulation and thereby disease outcomes (Kragesteen et

Mechanistic insights from skeletal disorders: Impacting the path to therapy

The ultimate hope for skeletal dysplasia patients is the availability of therapies that can ameliorate or prevent the dysmorphology. In recent years some progress has been made toward the development of therapeutic approaches for certain types of congenital dwarfism, based on the underlying mechanistic insights gained from fundamental research. Here, we highlight two examples for which mechanistic insights have been exploited and have entered human clinical trials: Achondrodysplasia and Schmid

Future directions and perspectives

Development of therapeutic approaches for congenital skeletal dysplasias requires knowledge of the lineage origins of skeletal cells and the properties of resident stem/progenitor populations in development, growth and disease. In recent years, lineage tracing experiments have revealed that a substantial fraction of hypertrophic chondrocytes survive and differentiate into osteoblastic cells during endochondral bone development and repair (Park et al., 2015; Yang, Tsang, Tang, Chan, & Cheah, 2014

Acknowledgments

K.C. and D.C. are supported by the Hong Kong Research Grants Council T12-708/12 N. We thank Tiong Tan for valuable comments on the manuscript and Wilson Chan for assistance with figure drawing.

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    Current address: ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.

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