Chapter 5 The Neural Crest and Craniofacial Malformations Hans J.ten Donkelaar and Christl Vermeij-Keers 5.1 Introduction the head (Vermeij-Keers 1990; Sulik 1996; LaBonne and Bronner-Fraser 1999; Le Douarin and Kalcheim The neural crest is a temporary embryonic structure 1999; Sperber 2002; Knecht and Bronner-Fraser 2002; that is composed of a population of multipotent cells Francis-West et al. 2003; Santagati and Rijli 2003). In that delaminate from the ectoderm by epitheliomes- addition, during later developmental stages multiple enchymal tranformation (EMT; Duband et al. 1995; places of EMT are recognized as well. In the head– Hay 1995; Le Douarin and Kalcheim 1999; Francis- neck area, for example, the neurogenic placodes and West et al. 2003). Neural-crest-derived cells are called the optic neural crest are such areas. Neurogenic pla- mesectodermal or ectomesenchymal cells (mesoder- codes, specialized regions of the embryonic ecto- mal cells of ectodermal origin) that have arisen derm, are the major source of primary sensory neu- through EMT. The neural crest was first described rons in the head (Johnston and Bronsky 1995; Gra- by His (1868) in the chick embryo as a Zwischen- ham and Begbie 2000). The vasculature of the head is strang, a strip of cells lying between the dorsal ecto- derived from mesoderm-derived endothelial precur- derm and the neural tube. Classic contributions in sors,while the neural crest provides the pericytes and amphibians identified interactions between tissues smooth muscle cells of the vessels of the face and the that lead to neural crest formation, and were re- forebrain (Etchevers et al. 2001). viewed by Hörstadius (1950). Cell labelling tech- Deficiencies in migration, proliferation and differ- niques, particularly the quail-chick chimeric marker entiation of neural-crest-derived tissue account for a (Le Douarin 1969, 1973), showed that the neural crest wide range of craniofacial malformations, i.e. the so- contributes to a large number of structures in the called neurocristopathies, manifested in a variety of avian embryo (Le Douarin and Kalcheim 1999; Le syndromes (Jones 1990; Gorlin et al. 2001; Wilkie and Douarin 2004),including the spinal,cranial and auto- Morriss-Kay 2001; Johnston and Bronsky 1995, 2002; nomic ganglia, the medulla of the adrenal gland, the Cohen 2002). Abnormalities in form, function or melanocytes and many of the skeletal and connective apoptosis of neural crest cells may range from von tissues of the head. The whole facial and visceral Recklinghausen’s neurofibromatosis through Treach- skeleton and part of the neurocranium are formed er Collins to DiGeorge and Waardenburg syndromes from the neural crest. Many species-related discrep- (Dixon et al. 2000). Neurocristopathies may be ac- ancies are present in the literature on neural crest cell companied by developmental disorders of the CNS. migration and their targets. In contrast to chick em- Recently, however, the idea that neural crest abnor- bryos, the neural crest in mammalian embryos is a malities underly the pathogenesis of the DiGeorge less distinct structure and can be defined as the tran- and Treacher Collins syndromes has been challenged sition zone between the neuroectoderm and the pre- (Sect. 5.5). Major craniofacial malformations are also sumptive epidermis from neural plate stages onwards found in holoprosencephaly (HPE) and the cran- (O’Rahilly and Müller 1999). In presomite murine iosynostoses. Holoprosencephaly is an early disorder embryos, the whole ectoderm, including the pre- of pattern formation that may lead to closely related sumptive neural crest, is able to produce mesectoder- forebrain and facial malformations. In the fetal peri- mal cells (Smits-van Prooije 1986; Smits-van Prooije od,craniosynostoses are frequent (approximately one et al. 1988). These EMT cells proliferate shortly after in 2,500 children) craniofacial malformations due to their migration into the mesodermal compartment agenesis or premature ossification of the cranial su- (Vermeij-Keers and Poelmann 1980). In somite tures, caused by mutations in FGFR and other genes stages during the transformation of the cranial neu- (Wilkie 1997; Cohen and MacLean 2000; Gorlin et al. roectoderm of the head folds via the neural groove 2001; Jabs 2002),that may interfere with normal brain into the neural tube, there is a balance between the development to varying degrees. outgrowth of the neuroectoderm and the production In this chapter, the neural crest and its derivatives of EMT cells by the neural crest; therefore, in mam- and craniofacial development will be discussed, fol- malian embryos, only short-distance migration of lowed by an overview of the neurocristopathies, HPE EMT cells occurs. and abnormal development of the skull, leading to The cranial neural crest provides the precursors CNS malformations.The neuropathology of HPE will of cartilage, bone, muscles and connective tissue of be discussed in Chap. 9. 192 Chapter 5 Craniofacial Malformations 5.2 Induction of the Neural Crest Neural crest cells are induced at the border between the neuroectoderm and the non-neural or surface ec- toderm (Fig. 5.1). During the formation of the neural tube in the chick embryo, neural crest progenitors come to lie in or directly adjacent to the dorsal neur- al tube (Le Douarin and Kalcheim 1999). Depending on the species, neural crest cells leave the neuroep- ithelium before, during or after neural tube closure, and ‘migrate’ throughout the body. To leave the neu- roepithelium, neural crest cells must lose their ep- ithelial characteristics and take on the properties of migratory mesenchymal cells. Wheat germ agglu- tinin labelling experiments in mouse embryos (Smits-van Prooije et al. 1988) and 1,1’-dioctadecyl- 3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) labelling in chick embryos (Kulesa and Fraser 1998) have shown that migration of neural crest cells is random. Other studies have suggested that there are cell-free spaces underneath the neuroectoderm and surface ectoderm resulting in specific pathways for migrating neural crest cells (Le Douarin 1969, 1973). In quail-chick chimeras, however, cell-free spaces may be created artificially.More recent studies suggest a role for chemoattractive signals such as fibroblast growth factor (FGF) in the control of neur- al crest cell migration (Kubota and Ito 2000; Francis- West et al. 2003). Induction of the neural crest appears to be a com- plex multistep process that involves many genes (LaBonne and Bronner-Fraser 1999; Aybar and May- or 2002; Knecht and Bronner-Fraser 2002; Gammill and Bronner-Fraser 2003; Wu et al. 2003). The gener- ation of neural crest cells appears to result from in- ductive interactions shared with the neural plate and the early epidermis. The neural–epidermal boundary can be distinguished by the expression of molecular markers such as the transcription factors of the Snail family, Snail and Slug, in Xenopus, zebrafish, chick Fig. 5.1 The early development of the human neural crest in and mouse (Mayor et al.1995; Sefton et al.1998; Link- a Carnegie stage 10 embryo.At rostral levels (a),crest material er et al. 2000; Aybar et al. 2003). The timing and ex- is formed before closure of the neural groove, whereas at more caudal levels (b–d) closure of the neural folds preceeds pression pattern of these markers differs between migration of crest material. (After Müller and O’Rahilly 1985) vertebrates. Bone morphogenetic protein (BMP) sig- nalling specifies the formation of dorsal neural tis- sues and the neural plate border (Sasai and De Robertis 1997; Weinstein and Hemmati-Brivanlou (LaBonne and Bronner-Fraser 1998; Gammill and 1999). Low or absent BMP signalling leads to neural Bronner-Fraser 2003; Wu et al. 2003). induction, whereas a high level of BMP signalling The epitheliomesenchymal tranformation (EMT) induces epidermis.An intermediate level of BMP sig- of emerging neural crest cells is accompanied by the nalling may determine the neural plate border (Wil- expression of the zinc-finger transcription factor son et al. 1997; Nguyen et al. 1998, 2000). In zebrafish, Slug. In chick and Xenopus embryos, its expression is BMP signalling is required for the development of the maintained during the phase of crest cell migration neural crest (Nguyen et al. 2000), but in Xenopus (Nieto et al. 1994; LaBonne and Bronner-Fraser intermediate BMP levels alone cannot induce the 2000). Slug mutant mice, however, do not show de- neural crest. Additional factors, Wnt proteins in par- fects in either neural crest or mesodermal tissues ticular, are required to induce the neural crest (Jiang et al. 1998). In mice, another family member, 5.3 Derivatives of the Neural Crest 193 Snail, rather than Slug is expressed in the regions un- Table 5.1 Derivatives of neural crest cells (after LeDouarin dergoing EMT (Cano et al. 2000; Locascio and Nieto and Kalcheim 1999; Sperber 2001) 2001; Knecht and Bronner-Fraser 2002; Gammill and Connective tissues Ectomesenchyme of facial promi- Bronner-Fraser 2003). Snail mutant mice die at gas- nences and pharyngeal arches trulation as a result of defects in mesoderm forma- Bones and cartilage of facial tion arising from deficient EMT. and visceral skeleton Dermis of face and ventral aspect of neck 5.3 Derivatives of the Neural Crest Stroma of salivary, thymus, thyroid, parathyroid and pituitary glands The neural crest is the major source of mesenchymal Corneal mesenchyme cells in the head and neck, and in addition gives rise Sclera and choroid optic