Our understanding of the genetic and molecular control of development in vertebrates has dramatically increased during the last 10 years through the discovery that molecular processes that control development in invertebrates have been conserved during evolution and are also found in vertebrates. Important developmental genes were identified that are not only similar in sequence but also in their molecular function in widely diverged organisms such as C.elegans, Drosophila, mice and man. It is now clear that epigenetic development is regulated by cascades of gene expression. Early acting regulatory genes initiate the developmental process and induce the expression of other downstream genes.
Phenotypic analysis of Drosophila mutants has allowed identification in the early eighties of more than 50 developmental genes that fall into three broad classes:
Classical embryology of the three types of skeletal elements originating from three distinct embryonic lineages will be described here and the involvement of several signaling factors in the regulation of skeletal morphogenesis will be discussed.
The ventral portion of the sclerotome surrounds the notochord and forms the rudiment of the vertebral body. The dorsal portion of the sclerotome surrounds the neural tube and forms the rudiment of the vertebral arches.
Segmentation and differentiation of the somitic subunits is under the control of regulatory genes and growth factors that act through specific inductive mechanisms.
The primary signal for sclerotome induction appears to be a notochord-produced factor called Sonic hedgehog (Shh). Pax 1 and Pax 9 are involved in mediating interactions between the notochord and the developing sclerotome.
Two basic helix-loop-helix transcription factors Twist and Scleraxis are also expressed in the sclerotome.
Appropriate differentiation of cervical, thoracic lumbar and sacr al vertebrae has been demonstrated to require sequential Hox gene expression. In human the Hox gene complex, homologous to the HOM-C complex in Drosophila, comprises 39 genes organized in four different chromosomal clusters (A, B C. and D). The genes are divided in 13 paralogous families based on protein sequence similarities and genes located at the 3?end of the cluster are expressed earlier during embryogenesis than their 5? neighbours. Although extensive overlap of function seems to exist among Hox genes, mutation induction in mice have provided relevant information as to their role. A null mutation of Hoxc-8 lead to transformation of the first lumbar vertebra into a 14th thoracic vertebra, and the eight rib became attached to the sternum. Loss of function alleles of Hoxb-4, Hoxa-2 and Hoxd-3 also induce vertebral transformations supporting the idea that Hox genes are responsible for modifying a common vertebral module thereby defining the identity of each vertebrae.
Cells from the lateral plate mesoderm (LPM) and cells from the lateral edges of nearby somites migrate to the presumptive limb field. The limb buds appear as small bulges protruding from the lateral body wall. Each limb bud consists of a mesenchymal core of mesoderm covered by an ectodermal cap. The ectoderm at the tip of the bud thickens to form a specialized structure called the apical ectodermal ridge (AER).
This structure maintains continuous limb bud outgrowth along the proximo-distal (P-D) axis (shoulders to digits). Concomitant to its elongation along the P-D axis, the limb becomes flattened along the dorso-ventral (D-V) axis (back of hand to palm) and asymmetric along the antero-posterior (A-P) axis (thumb to little finger). The most proximal elements (stylopod) begins to differentiate first, followed by the progressive differentiation of more distal structures (zeugopod and autopod). This outgrowth and patterning depends on the establishment and maintenance of three signaling centers within the limb bud:
The limb buds in vertebrates grow with respect to the proximodistal, dorsoventral and craniocaudal (antero-posterior) axes and require positional signaling molecules.
The FGF family comprises at least 24 members. The proteins encoded by the 24 different genes are variable in length (155-268 amino-acids) and contain a conserved "core&" sequence of ~ 120 amino-acids that confers ability to bind heparin or heparan sulfate proteoglycans (HSPG). Secreted FGF are able to bind to HSPG (low affinity receptors) such as syndecans, glypican and perlecan located at the cell surface which restrict their ability to diffuse far from the cells. This also allows their binding to high affinity receptors, the Fibroblast Growth Factor Receptors (FGFRs) which form a family of 4 transmembrane protein tyrosine kinases. The binding of FGF to monomers of FGFR induces receptor dimerization and activates their tyrosine kinase activity that itself triggers signal transduction. At least five FGF (FGF 2, FGF 4, FGF 8, FGF 9 and FGF 10) and two FGFR (FGFR1 and FGFR2) are expressed during limb bud initiation.
FGFs produced in the AER serve at least two major functions:
One of the target for FGF signaling from the AER is FGF 10 which is expressed in the distal limb bud mesenchyme. This factor is able to interact with FGF 8 and there might be a positive feed-back loop between FGF 10 and FGF 8. This reciprocal regulation is likely to be mediated by two isoforms of FGFR 2, FGFR 2b (that binds FGF 10 exclusively) and FGFR 2c (that binds FGF 8). A recent model has been proposed in which FGF 10 made in the mesenchyme of the limb field diffuses in the ectoderm where it binds FGFR 2b and induces FGF 8 in the ectoderm. The FGF 8 in turn diffuses into the mesoderm and activates FGFR 2c which causes the upregulation of FGF 10. The FGF 10 then continues the loop and results in limb bud induction.
Hence FGFR 2 appears to be essential for limb bud initiation whereas FGFR 1 seems to play an essential role at several stages of limb development. This assertion is based on the study of mouse models and expression patterns which have revealed an important function of FGFR 1 in specification of P-D axis formation. FGFR 1-mediated signals are required for maintaining ZPA and progress zone activities.
Some of the FGF in conjonction with Shh can affect expression of the bone morphogenetic protein (Bmp-2 and 7) and Hox genes, mostly Hoxd-12 and Hoxd-13. These latter genes are members of the Hoxd complex and are expressed within the distal wrist (Hoxd 12) and within the hand and fingers (Hoxd 12 and 13). The role of the Hoxd 13 gene in the proximodistal differentiation of limb segments has been illustrated by the demonstration that mutations in the human gene transforms the metacarpals to carpals and metatarsals to tarsals.
According to a process called endochondral ossification, the skeletal elements of the limb develop from a column-like mesodermal condensation that appears along the long axis of the limb bud. Mesenchymal cells condense to form prechondrogenic. Prechondrocytes in the prechondrogenic condensations differentiate into chondrocytes in response to growth factors and secrete molecules characteristic of the extracellular matrix such as collagen type II and aggrecan (a large proteoglycan). The initial phase of chondrification results in the formation of a cartilaginous envelope, the perichondrium. This perichondrium in which bone morphogenetic protein 2, 4 and 7 (BMP 2, 4 and 7) and parathyroid hormone/ parathyroid hormone-related peptide receptor (PTH/PTHrPR) are expressed, inhibits chondrocyte proliferation and maturation thereby helping to control the growth and differentiation of the forming cartilage elements.
As the cartilage elements grow different zones can be distinguished that demarcate the progressive differentiation of the chondrocytes.
Defective cartilage growth occurs in a wide spectrum of disorders called chondrodysplasias that usually result in dwarfisms of variable severity. The most common of these disorders is achondroplasia , a dominant genetic disease caused by a recurrent activating mutation in the transmembrane domain of FGFR3 affecting chondrocyte proliferation and differentiation.
The skeleton of the head in human is made up of chondrocranium (neurocranium), membrane bones and viscerocranium.
The skeletal elements of the pharyngeal arches are derived from neural crest and lateral plate mesoderm. Proper development of the pharyngeal arches relies on the expression of Hox genes. Gene inactivation of Hoxa-2 result in the replacement of the second pharangeal arch by a duplicated set of proximal first pharangeal arch elements. Hence Hoxa-2 normally permits only endochondral ossification to occur in the second arch whereas both endochondral and membranous ossifications take place in the first arch. Other factors are implicated in the differentiation of the pharangeal arches including Prx1 and 2 (two closely related paired class homeobox genes), and homeodomain proteins of the Dlx family (Dlx 1, 2, 3, 5 and 6. Dlx genes could specify regional fate of the pharangeal arch ectomesenchyme. Mice lacking Dlx 5 showed craniofacial abnormalities with delayed ossification of the calvaria suggesting multiple roles of this gene in branchial (pharangeal) arches.
The signaling pathways acting to regulate calvarial growth and cranial suture morphogenesis are also beginning to be elucidated. They involve different transcriptions factors including MSX1 and 2 (two Msh-like homeobox genes), Shh, BMPs (BMP2 and 4), TGFb and Twist but also tyrosine kinase receptors FGFRs and their ligands FGFs.
The importance of FGF/FGFR signaling in human skull development has been revealed by the demonstration that premature fusions of the sutures that produce craniosynostoses (Apert, Crouzon and Pfeiffer syndromes being the most common) are often caused by mutations in the FGFR genes. Most mutations are found on FGFR 2 that account for most if not all cases of Apert and Crouzon syndromes. Mutations in FGFR 1 and FGFR 3 have been identified in some cases of syndromic craniosynostoses (Pfeiffer and Muenke syndromes). Most of these mutations would induce ligand independent activation of the receptor but could also alter ligand specificity. Thus FGFR appear to play a key role in the proliferation-differentiation of osteogenic stem cells in the fetal coronal sutures. FGFR2 would be expressed in proliferating cells whereas the onset of differentiation would be accompanied by up-regulation of FGFR 1. Craniosynostosis can also be caused by mutations in MSX 2. Both MSX1 and MSX2 are expressed in the sutural mesenchyme and induced by BMP 4 and similarly to mice deficient for the FGFR 2(IIIb) isoform, Msx 1 deficiency is associated to defective mandible while disruption of the Msx 2 gene result in a central defect of the frontal bone. Thus correct dosage of Msx 2 seems to be critical for normal osteogenic differentiation in the mammalian skull. Altogether it appears that conserved signaling pathways including BMPs, Msx and FGF/FGFRs regulate tissue interactions during suture morphogenesis and intramembranous bone formation of the calvaria. The involvement of FGFRs in both craniosynostosis and limb abnormalities (achondroplasia, thanatophoric dysplasia) support the idea that craniofacial and limb development utilize common signaling pathways which, however, remain to be clearly elucidated in human.
Great progress has been made recently in understanding limb development which appears as one of the best model of morphogenesis. Nevertheless the specific target genes regulated by Hox genes that influence growth and patterning of skeletal elements remain unknown. Important clues have been provided by the recent demonstration that secreted signaling molecules (FGFs, BMPs, Wnts, Shh) restrict their own activities by inducing antagonists in their responsive cells. Hence activation of the Shh/FGF loop could result from competing combinatorial associations of successively expressed distal Hox proteins with Meis-Pbx complexes. In the coming years, complementary approaches including surgical manipulations, ectopic expression studies as well as targeted gene disruption should provide new insights into the mechanisms of skeletal development.
Atlas of Genetics and Cytogenetics in Oncology and Haematology 2001-09-01
Skeletal Development in Human
Online version: http://atlasgeneticsoncology.org/teaching/30075/skeletal-development-in-human