If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down. But I can find out no such case.
Charles Darwin (1859)
I never forget a face, but in your case I'll make an exception. Groucho Marx
Approximately one hundred years separate the above quotations but both are salient observations on the importance of the uniqueness of the human face. The face is the most distinguishing feature of the vertebrate body as can be seen in the six billion unique faces of the world's human population. Advances in craniofacial biology demonstrate that “successive, slight modifications” in signaling pathways have resulted in the kind of diversity seen in the animal kingdom noted by Darwin, the humorous countenance that prompted the remark by Groucho and, at the same time, the extreme variations that result in craniofacial pathology [Brugmann et al., 2006]. It has been some 350 million years since the first animal crawled out of the sea and onto the land. Ahlberg et al. 2005 recently demonstrated that this animal, Ichthyostega, might not have actually crawled but rather inched its way over the land, probably dragging its hind legs and long tail behind. This creature, with very short legs and long tail, may have propelled itself by arching its spine and propelling itself forward like an inchworm. Nonetheless, Ichthyostega possessed the already ancient tetrapod body form of paired fins/limbs and vertebrate head.
An understanding of the evolution of the head and the finely tuned temporospatial signaling pathways involved is critical to understanding the origins of the vertebrates as well as of human craniofacial malformations. Congenital anomalies are a major cause of infant mortality and childhood morbidity, affecting 2–3% of all babies. Approximately 1% of these newborns have syndromes or multiple anomalies; craniofacial anomalies are often a component part. In fact, three-fourths of human birth defects have malformations involving the craniofacial region (see Centers for Disease Control, Birth Defects Research and Prevention at http://www.cdc.gov/ncbddd/bd/centers.htm, in press). Craniofacial anomalies, other than cleft lip and palate, occur in 1 in every 1,600 newborns in the United States of America and include jaw deformities, malformed or missing teeth, defects in the ossification of facial or cranial bones, and facial asymmetries [WHO, 2001].
The bony skull is formed from two components, the neurocranium and the viscerocranium, and comprises 22 separate bones, 20 deciduous and 32 permanent teeth. The whole viscerocranium and part of the neurocranium are formed from the neural crest. The elaboration of the neural crest probably represents the most important step in the evolution of the vertebrate head. Cranial neural crest precursors were present in the common ancestors of all extant chordates including vertebrates [Wilkie and Morriss-Kay, 2001]. The most primitive vertebrates did not have jaws. Though the paired fin/limb evolved shortly before jaws there is remarkable overlap of the signaling pathways co-opted in head and limb development; so much so that genes involved in limb development are good candidates for genes that might be involved in errors of craniofacial morphogenesis.
Most of the tissues and organs of the craniofacial region are derivatives of neural crest cells (NCCs). NCCs populate the frontonasal process, first, second, third, and fourth pharyngeal arches and contribute to neural, skeletal, dermal, and mesenchymal structures. Because of this pluripotency, the neural crest has been referred to as the fourth germ layer [Hall, 2000]. In this issue of the journal Cordero et al. 2010 discuss the tightly regulated spatial and temporal signaling network required for the induction, migration, proliferation, and differentiation of NCCs that are the primordia of craniofacial structures. The many aberrations in the complex multi-step processes that result in craniofacial malformations are described. They next demonstrate the unique interactions of individual populations of NCCs that populate the seven prominences that comprise the vertebrate face with adjacent cells required for normal craniofacial morphogenesis. Examples of human disorders during the hierarchical progression of development are described. A section is devoted to exciting future areas of investigation including the use of the next generation of sequencing and hybridization technologies, advances in NCC culture systems, the role of micro RNA in NCC development and, finally, epigenetic regulation of neural crest fate.
NCC patterning within the pharyngeal arches is dependent upon a number of signaling pathways including bone morphogenetic proteins (Bmps), fibroblast growth factors (Fgfs), and retinoic acid (RA) [Helms et al., 2005]. The endothelin family was first described in 1988 with the cloning of endothelin-1 identified by its potent vasoconstrictive activity [Yanagisawa et al., 1988]. In addition to its role in dynamic regulation of blood pressure, endothelin signaling plays an important role in NCC patterning [Baynash et al., 1994; Hosoda et al., 1994]. For example, mutations in human enteric neurons of the EDNRB gene are responsible for some cases of Hirschprung disease. The review by Clouthier et al. 2010 demonstrates that endothelin signaling is arguably the key mediator of NCC development and skeletal patterning in the mandibular arch. This family of ligands and receptors is highly conserved across vertebrates [Clouthier and Schilling, 2004] and appears to function by inducing factors required to establish the identity of ventral (distal) NCCs in the mandibular arch while repressing signals associated with the identity of more dorsal (proximal) NCCs.
Retinoic acid, its nuclear receptors and the enzymes required for its synthesis are endogenous to the developing inner ear, suggesting that retinoic acid is part of a critical signaling pathway especially indispensable for inner ear development [Biesalski and Weiser, 1989; Romand et al., 2002, 2006]. However, the normal function of retinoic acid is achieved only at optimal homeostatic concentrations and an excess or deficiency results in inner ear dysmorphogenesis. Frenz et al. 2010 contribute original research findings that illustrate that retinoic acid coordinates inner ear morphogenesis by controlling an FGF/Dlx signaling cascade where deviations in local retinoid concentrations lead to inner ear dysmorphogenesis. The effect of too little or too much retinoic acid on FGF/Dlx signaling is described as the “Goldilocks phenomenon.”
BMP and Wnt signaling pathways are required for normal development of all ectodermal appendages, including teeth in rodents and humans. Ohazama et al. 2010 identify a novel modulator of extracellular signaling in craniofacial organogenesis. They demonstrate an interaction between an extracellular protein (Wise) that combines Bmp ligands and a low-density lipoprotein receptor (Lrp4) that acts to link these two pathways in a novel manner. Wise and Lrp4 together act as an extracellular mechanism of coordinating BMP and Wnt signaling. The integration of these two pathways is essential for the regulation of the development of craniofacial organs, especially teeth, through epithelial–mesenchymal interactions.
Recent advances in our understanding of the primary cilia has shed light on the pathogenesis of some craniofacial disorders. Oro-facial-digital (OFD1), Meckel–Gruber (MKS), Joubert and Ellis-van Creveld syndromes are all established ciliopathies. The primary cilium is a ubiquitous organelle which facilitates interactions between the cell and its environment much like signal transduction pathways. Brugmann et al. 2010 illustrate why craniofacial disorders can be usefully classified as ciliopathies in order to elucidate the underlying molecular defects. For example, experimental evidence exists that shows that ciliary dysfunction results in abnormal Wnt and Hedgehog signaling.
The treatment of the more than 700 recognized disorders with significant craniofacial malformations requires the integrated interventions of, among others, surgeons, dentists, audiologists, geneticists, and psychologists, treating patients sometimes over many years and the outcomes are often variable and unsatisfactory. Treacher Collins syndrome (TCS) is a severe disorder of craniofacial development caused by mutations in the TCOF1 gene. The cellular basis for TCS is a deficiency of NCCs secondary to extensive p53 dependent neuroepithelial apoptosis [Dixon et al., 2006]. Treatment of Tcof1+/− embryos (the mouse model of TCS) with a specific inhibitor of p53 results in the rescue of cranioskeletal development. A more efficient rescue was observed when p53 was blocked genetically [Jones et al., 2008]. Trainor 2010 describes the prevention of TCS craniofacial anomalies as the first successful rescue of a congenital neurocristopathy which may provide a model for future research in craniofacial defects with a similar etiology and pathogenesis.
Finally, Melville et al. 2010 present a challenging image of future genetic and pharmacological treatment approaches of craniosynostosis. Current treatment options are almost exclusively surgical with the risk of complications in infants requiring either open or endoscopic repair. The young age of intervention, between 3 and 12 months, increases the risks for infection, seizures, bleeding and optical nerve ischemia. The majority of cases of syndromic craniosynostosis are caused by mutations in a relatively small number of genes including the fibroblast growth factor receptor (FGFR) family of proteins, RAB23, EFNB1, TWIST, and MSX2. A convergence of mutations in aberrant FGFR signaling has been found in various cancers and craniosynostosis [Wilkie, 2007]. These are promising targets for pharmacological and gene therapy in the future. An exciting possibility is the use of small molecule inhibitors designed to target the FGFR2 and FGFR3 pathways, especially the activated FGFR tyrosine kinase, to rescue premature suture fusion. This has already been accomplished in mouse calvarial organ culture and efforts to utilize these inhibitors in vivo have been promising [Shukla et al., 2007]. Preclinical and clinical trials for treatment of cancer using these inhibitors are underway. Genetic strategies include infection of the posterior frontal suture in rats with a dominant-negative FGFR1 construct to prevent normally occurring fusion [Greenwald et al., 2001] and the use of RNA interference both in vitro and in vivo.
The development of the craniofacial complex, just as that of any part of a multicellular organism, depends upon the communication between cells mediated primarily by extracellular signal molecules. The long delay of about 2.5 billion years between the appearance of prokaryotes and multicellular organisms probably reflects the difficulty of evolving a language of cellular communication and their evolutionary centrality. At the same time, it is now clear that an understanding of the general principles of cell communication can be translated into the elucidation of the pathogenesis of craniofacial disorders and hopefully into therapeutic modalities. This series of articles demonstrates that the emerging field of craniofacial biology is attracting scientists from a wide range of disciplines including clinical geneticists, developmental and evolutionary biologists, dentists, pharmacologists, and surgeons. The future collaboration of members of these disciplines will add to our knowledge as to how the highly conserved molecular pathways and their functions in craniofacial development create a face and not a limb bud and enable new non-surgical options for the treatment and prevention of these complex disorders.