Molecular basis for skeletal variation: insights from developmental genetic studies in mice.

The axial skeleton develops through a series of complex processes that require the coordinate regulation of many cellular and molecular events (Gilbert, 1994; Kaufman and Bard, 1999). During gastrulation of the vertebrate embryo, the formation of the primitive streak and notochord represent the first step in the formation of axial and paraxial mesoderm. The paraxial mesoderm is then segmented into somites, which in turn are further subdivided into rostral and caudal halves. The somites are the origin of sclerotomal cells that migrate to form condensations of mesenchymal cells that produce the cartilaginous ‘models’ for the skeletal elements. Through chondrification and ossification, prevertebrae and bones of the axial skeleton grow and achieve their final size and shape. Recently, experimental studies in chicken and genetic approaches in the mouse have contributed a wealth of molecular markers for different stages of axial skeleton development, and at the same time provided evidence for the critical functional role of various types of molecules in the formation of the axial skeleton. The results from these studies are now beginning to be integrated into a molecular framework of gene regulation and cell differentiation during skeletal development. It is expected that this knowledge will help to better understand the etiology of skeletal variations, and provide a scientifically informed basis for their evaluation as study parameters for potential reproductive toxicants. Understanding the causes of variations in skeletal development is particularly important in light of the fact that skeletal variations are highly prevalent in the general population. Of 10922 asymptomatic young men whose axial skeleton was examined by X-ray, only 2.6 % resembled the textbook ideal and showed no pathological alterations of the axial skeleton (Hald et al., 1995). While 80% of axial abnormalities involved habit related mild scoliosis, the authors also reported more pronounced abnormalities in about 40–45% of subjects, including deformations of vertebral bodies and changes in vertebral identity. For many people, such abnormalities may have occupational implications, and may affect general health. In the mouse, skeletal abnormalities often do not interfere with viability and reproductive capability. This allowed the isolation and maintenance of mutants with skeletal abnormalities, which either arose spontaneously or were induced during radiation and mutagenesis experiments (Lyon et al., 1996). On the basis of phenotypic and embryological analyses, mutations could be grouped into different classes reflecting the different steps of skeletal development affected (Gruneberg, 1963). Positional cloning techniques now permit the isolation of the genes underlying observed defects as successfully demonstrated for the cases of Brachyury (T; (Herrmann et al., 1990), short ear (se; (Kingsley et al., 1992) and brachypodism (bp; (Storm et al., 1994). A complementary approach is to analyze the function of genes that are known to be expressed during formation of the axial skeleton. Many of these genes were thought to be required for normal skeletal development, based upon their similarity to developmental genes identified in mutant screens in Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). In the mouse, loss or gain of function mutants can be created through homologous recombination in embryonic stem cells and the subsequent production of mice completely derived from these cells (Joyner, 1993), and through transgenic approaches (Hogan et al., 1986). As the phenotypes of genetically manipulated mice resemble the malformations observed in a variety of human skeletal dysplasias, the mouse provides an excellent model system to understand the molecular and cellular pathways steps involved in human skeletal malformations. Finally, not only genetic but also environmental factors influence skeletal development. Interfering with one of the many steps involved can give rise to malformations that resemble those caused by known genetic defects. Such resemblances suggest that similar molecular and cellular pathways are affected in both cases, and may help to identify the molecular basis for the actions of exogenous substances. The most prominent examples for the skeletal system are the Hox genes, whose expression is altered by retinoic acid. In this way, exposure to an environmental factor produces phenotypes similar to those caused by mutation or manipulation of Hox genes themselves (Kessel, 1992). In this chapter, we will give a short overview of axial skeleton development in the mouse, focusing on selected morphological events. We will then review current knowledge on the molecular basis of skeletal development, including a discussion of genes that regulate growth and cell differentiation in skeletal elements. A section on selected environmental factors that interact with the complex genetic network controlling skeletal development will conclude this review.