it has been over a century since the discovery that migratory cells of the neural crest give rise to the craniofacial skeleton , unlike the vertebral and limb skeletons , which are derived from embryonic mesoderm . despite the extraordinarily high frequency of cleft palate and other craniofacial problems in human birth defects , the genetic control of this part of the skeleton however , recent identification of the genetic basis for many craniofacial syndromes , combined with functional studies in animal models , are beginning to illuminate the cellular and molecular mechanisms underlying skull develop ment and its origins in the neural crest . studies over the past few years , including one by schwend and ahlgren   published recently in bmc developmental biology , have taken advantage of the availability of zebrafish mutations that disrupt the skull to uncover genes that control its patterning in the early embryo . moreover , advances in the ability to create transgenic zebrafish have opened up new avenues for following neural crest cells in living embryos and thereby elucidating how genes control skull morphogenesis . a fundamental feature of the head is its modular organization - the embryonic hindbrain and the pharynx become partitioned into a series of segments . within 24 hours postfertilization the zebrafish hindbrain has become segmented into seven rhombomeres and the tissues surrounding the pharynx subdivide into seven so - called arches ( figure 1a ) . these include the mandibular arch ( arch 1 ) , which forms the jaw , the hyoid arch ( arch 2 ) , which forms the jaw support , and five gill arches ( arches 3 - 7 ) . all have a similar structure , including skeletal , neural and glial components derived from neural crest , muscles and blood vessels derived from mesoderm , and an outer sheath of surface ectoderm and endoderm . pharyngeal arch and neurocranial cartilage patterns in wild - type and hh - deficient zebrafish larvae . alcian - stained cartilages were dissected and flat mounted ; dorsal views are shown , anterior to the left . ( a ) the wild - type ( wt ) pharyngeal arches at 4.5 days postfertilization include : the mandibular arch ( arch 1 ) , containing meckel 's cartilage ( m ) and the palatoquadrate ( pq ) ; the hyoid arch ( arch 2 ) , containing the ceratohyal ( ch ) and hyosymplectic ( hs ) ; and five more posterior , branchial arches ( 3 - 7 ) , each containing a ceratobranchial cartilage . ( b ) the wild - type neurocranium at 4.5 days postfertilization includes paired trabeculae ( t ) and an ethmoid plate ( e ) . these similarities led early comparative anatomists to argue that jaws , which form from the prominent mandibular arch , evolved from an ancestral gill arch ( reviewed in ) . consistent with this idea , early vertebrates had pharyngeal arches but were jawless - arches are one of the most ancient vertebrate traits , found even in non - vertebrate chordates such as amphioxus . evidence to support the gill - arch origin for jaws has come with the discovery of similar patterns of gene expression in every arch in the series . for example , recent studies in a chondrichthyan fish , a skate , suggest that the network of secreted growth factors found in the mandibular arch , which includes sonic hedgehog ( shh ; see below ) , fibroblast growth factor and retinoic acid , is conserved in the gill arches , and inhibitors of shh signaling disrupt skeletal patterning similarly in every arch . surprisingly , however , recent genetic evidence argues against the model of a gill - arch ground plan and instead suggests that the default state for an arch is more mandibular in character . combinatorial expression of hox genes underlies arch - specific morphologies , with the exception of the mandibular arch , which is devoid of hox activity . this ' hox code ' applies to all non - mandibular arches ( there are four arches in mammals but up to nine in other vertebrates ) and in each case posterior arches acquire more anterior characteristics in the absence of hox gene function . for instance genetic knockout of hoxa2 in mice , or combined loss of hoxa2 and hoxb2 in zebrafish , leads to a replacement of second - arch - derived structures by those of the first arch . strikingly , minoux et al .   recently showed that all posterior arches take on a mandibular identity following the conditional loss of all hoxa cluster genes in mouse cranial neural crest cells . thus , posterior arch identity must override a default ' mandibular ' program that dominates in the absence of hox expression . once head segmentation is established , the next critical step is differential growth and patterning within each arch . in most vertebrates gill arches are prominent structures in aquatic vertebrates such as fish , but are only transiently present in mammals and later become incorporated into the throat and larynx . how does the identity acquired through the hox code result in such large differences in arch morphogenesis ? the answer appears to lie in how neural crest cells within the arch respond to signals from surrounding epithelia . early grafting studies in avian embryos demonstrated that mandibular neural crest transplanted posteriorly gave rise to an ectopic beak and other mandibular structures , suggesting that similar signals exist in both anterior and posterior arches , and that segmental identity is intrinsic to neural crest cells . more recent variations on these types of grafts suggest that this is only part of the story . nevertheless , further evidence for an intrinsic hox code in the neural crest has come from studies of zebrafish moz mutants , which disrupt an essential activator of hox paralog group 2 gene expression and exhibit transformations of the hyoid ( arch 2 ) to a mandibular ( arch 1 ) identity , similar to hoxa2/b2 double morpholino - injected ( morphants ) . mosaic studies combining mutant and wild - type cells suggest that moz and hox paralog group 2 proteins act cell - autonomously in the second arch skeletogenic neural crest cells - without them skeletal elements acquire the size and shape of their counterparts in the mandibular arch . thus , hox genes appear to instruct neural crest cells to interpret arch signals appropriately . these signals largely come from the endodermal and/or ectodermal epithelia that ensheath each arch . here again , mutational studies and transgenics in zebrafish have been very informative . time - lapse movies of fluorescent trans - genes that mark skeletogenic neural crest cells reveal that early cartilage formation occurs in close proximity to the endodermal ' pouches ' that separate adjacent arches - these are highly conserved from fish to humans . zebrafish mutants that lack endoderm fail to form cartilage , and restoring endoderm rescues skeletogenesis . mutants that disrupt individual pouches disrupt adjacent cartilages , presumably because of the loss of chondrogenic signals emitted by the pharyngeal endoderm . one secreted signaling molecule expressed by this endoderm and required for craniofacial development is shh , a vertebrate relative of the product of the drosophila segment polarity gene hedgehog . hh signaling influences multiple aspects of craniofacial development . in humans , disruption of shh , as well as several other components of the hh signal transduction pathway , causes holoprosencephaly ( hpe ) , which is among the most common craniofacial birth defects , characterized by cyclopia , midfacial clefting and arch defects . conversely , ectopic expression of shh in chick embryos causes complete duplications of the mandible . hh signaling impinges on multiple cell types , including pharyngeal endoderm , surface ectoderm , ventral brain and neural crest . much of what is known centers around the midline - fusion or loss of midfacial bones are typical of hpe patients , as well as of shh - deficient mice or zebrafish ( figure 1b , c ) . new insights came with the knockout of the essential hh co - receptor , smoothened ( smo ) , which when removed specifically from neural crest cells causes similar craniofacial defects in mice as the knockout . many outstanding questions remain , however , including the location of the sources of shh and its relatives in the face , the precise timing of their actions , and how defects in hh signaling cause such variable defects ( for example , midline fusions versus cleft palate ) . here again , zebrafish offer the advantage of a forward genetic approach and a large collection of mutants in the hh signaling pathway . fate - mapping studies in both shh and smo mutants , using transgenics that label migrating cranial neural crest cells , have revealed that hh signaling plays two distinct roles in patterning the cartilages of the primary palate : one in neural crest cells in the maxillary region of the first arch , and the second in a more anterior population of neural crest cells that forms the ethmoid plate in the midline . disruption of hh signaling leads to defects in one or both populations , resulting in cyclopia ( when maxillary regions fuse ; figure 1c ) or mid - facial clefting ( when the midline ethmoid is not induced ) , and treatment with the alkaloid cyclopamine , which chemically blocks smo function , only disrupt one or the other of these populations , depending on the stage of treatment . shh expressed in the ventral forebrain and oral ectoderm appears to be the critical source in the midline . these results exemplify the complex spatial and temporal nature of hh signaling in the face , and help explain why human hpe manifests itself in so many different ways . hh signaling also appears to have distinct roles in the mandibular and more posterior arches . schwend and ahlgren   show that chameleon ( con ) mutants , which lack a fully functional dispatched ( disp1 ) protein crucial for hh secretion , develop maxillary fusions , loss of jaw joints and a complete lack of cartilage in arches 3 - 7 . disp1 co - localizes with shh in pharyngeal epithelia shortly after neural crest migration into the arches . defects in the mandibular arch are not surprising , though the results in this paper hint at a previously unappreciated role for hh signaling in jaw joint development . what is more surprising is the selective loss of expression of some genes involved in skeletogenesis ( sox9a , dlx2a ) and not others ( for example , sox9b , hand2 ) in the gill arches in con / disp1 mutants . in zebrafish , defects in the gill arches are often secondary consequences of developmental delay or heart defects . however , this is clearly not the case in con / disp1 mutants and instead , fibrous connective tissue appears to replace cartilage . thus , hh signaling is required for cartilage differentiation in the posterior arches , and several lines of evidence suggest that this is distinct from its roles in the palate or mandible . such specific regulation of gene expression and skeletogenesis by hh may offer insights into the nature of defects in other zebrafish mutants that affect the posterior arches . given that shh and other signals are expressed in similar patterns in different arches , one big open question is how the responses to these signals are modulated in a segment - specific manner . we still know relatively little about the targets of hox genes within different segments , or how the combinatorial hox code is interpreted to give different morphologies . what seems clear is that not all cells within a segment necessarily interpret the code in the same fashion , nor do the signals impinging upon them have a single effect on cell behavior . for example , shh appears to have at least two roles in the face , one in establishing the patterns of palate or joint precursors and one in cartilage differentiation , both in the palate and in the gills . whether this is truly a dual role for shh or reflects distinct roles for its relatives , such as indian hedgehog ( ihh ) , zebrafish provide the opportunity to address these questions more directly than is possible in a chick or mouse , as we can watch neural crest and arch development in real time using transgenics , taking advantage of the transparency of the live fish embryos . the added ability to screen for craniofacial mutants in zebrafish is proving fruitful for discovering novel factors involved in arch development . the importance of this approach is apparent when considering that craniofacial defects are so common ( for example , 1 in 700 live births have cleft lip or palate ) , yet the genetic basis for most is unknown . most of the genes known to be involved have roles in embryonic development , arguing that as new genetic causes are revealed , their functions can be rapidly evaluated in the context of the known pathways that pattern the arches . craniofacial research is now undergoing a rapid expansion , with the accelerated identification of human disease genes and new model systems for functional analysis , and the zebrafish promises to be a central player .
zebrafish are a powerful system for studying the early embryonic events that form the skull and face , as a model for human craniofacial birth defects such as cleft palate . 
 signaling pathways that pattern the pharyngeal arches ( which contain skeletal precursors of the palate , as well as jaws and gills ) are discussed in light of a recent paper in bmc developmental biology on requirements for hedgehog signaling in craniofacial development.see research article http://www.biomedcentral.com/1471-213x/9/59