Zebrafish Developmental Models of Skeletal Diseases

Abstract

The zebrafish skeleton shares many similarities with human and other vertebrate skeletons. Over the past years, work in zebrafish has provided an extensive understanding of the basic developmental mechanisms and cellular pathways directing skeletal development and homeostasis. This review will focus on the cell biology of cartilage and bone and how the basic cellular processes within chondrocytes and osteocytes function to assemble the structural frame of a vertebrate body. We will discuss fundamental functions of skeletal cells in production and secretion of extracellular matrix and cellular activities leading to differentiation of progenitors to mature cells that make up the skeleton. We highlight important examples where findings in zebrafish provided direction for the search for genes causing human skeletal defects and also how zebrafish research has proven important for validating candidate human disease genes. The work we cover here illustrates utility of zebrafish in unraveling molecular mechanisms of cellular functions necessary to form and maintain a healthy skeleton.

Introduction

Over the last few decades, zebrafish (Danio rerio) has been used primarily to further our understanding of developmental processes and to discover genes required for development through the use of unbiased phenotype-driven forward genetic screens (Driever et al., 1996, Haffter et al., 1996, Howe et al., 2013, Knapik, 2000). In recent years, the use of zebrafish model has been expanded to address a repertoire of clinically defined disorders (Haesemeyer and Schier, 2015, Kokel et al., 2010). This trend is sustainable because zebrafish offers a compromise between physiological complexity of a mammalian vertebrate and morphological simplicity of invertebrates, along with a comprehensive toolbox of cell biological (Unlu et al., 2014, Vacaru et al., 2014) and genetic tools at a modest cost relative to mammalian models. Within the transparent body of zebrafish, morphogenetic processes and physiological activities of the skeleton are easily accessible, and the chondrocytes and osteocytes can be labeled with simple stains, allowing for rapid and high-throughput assessment of the skeleton.

As vertebrates, the zebrafish and mammalian skeletons are very similar. Although mechanics of the mammalian (terrestrial) and fish (aquatic) skeletons differ due to their habitats (Kimmel et al., 2001, Miyashita, 2016, Witten and Huysseune, 2009), their similarities are strongest at the level of basic cellular functions of chondrocytes and osteocytes and their primary role of secreting extracellular matrix (ECM). The ECM is a dynamic structure that provides not only a mechanical context to cells, but it also maintains gradients of growth factors and morphogens necessary for homeostasis, proliferation, and differentiation of progenitor cells to mature tissue. The components of ECM, including bound water, proteins, and polysaccharides, modulate diffusion of signaling factors and provide structural support and scaffolding for developing tissues.

Forward genetics screens have been especially useful in identification of novel skeletal mutations in zebrafish (Andreeva et al., 2011, Neuhauss et al., 1996, Nissen et al., 2006). With forward genetic approaches and positional cloning methods, novel zebrafish models have been identified to study hereditary skeletal disorders. Availability of microsatellite-based genetic linkage maps afforded fast and reliable positional cloning to identify mutated genes in zebrafish (Fornzler et al., 1998, Knapik et al., 1996, Knapik et al., 1998). Genetic linkage analysis is a mainstay of disease gene identification, and additional methods such as SNP-based genetic maps and whole-exome sequencing approaches have enhanced robustness of the zebrafish model (Bradley et al., 2007, Guryev et al., 2006).

Zebrafish as a model organism offers multiple routes of gene depletion strategies including classical chemical mutagenesis with N-ethyl-N-nitrosourea (ENU) (Mullins et al., 1994, Solnica-Krezel et al., 1994), insertional mutagenesis (Amsterdam et al., 2004), gene knockdown via morpholino-oligonucleotide (MO) injections (Nasevicius & Ekker, 2000), and newer techniques such as zinc-finger nuclease (Doyon et al., 2008), TALEN (Huang et al., 2011), and CRISPR/Cas9 genome editing systems (Hwang et al., 2013, Jao et al., 2013).

Here, we focus on the primary functions of the skeletal cells to synthesize, secrete, and modify proteins of the ECM and how disruptions of these highly conserved metabolic processes in the zebrafish model have informed our understanding of human skeletal pathology.