Developmental Biology and Morphogenesis of the Face, 1 Lip and Palate Alphonse R. Burdi

Prologue. The “history of man for the nine months preceding his birth would, probably, be far more interesting and contain events of greater moment than all the three score and ten years that follow it.”

Samuel Taylor Coleridge, Miscellanies, Aesthetic and Literary. Circa 1800.

The developmental biology of the face, lip, and palate active behaviors at intracellular, cell surface, and ex- is best understood against a backdrop of biological tracellular matrix levels. Complete or partial inter- paradigms and information drawn from the multidis- ruptions of any one or combination of these phenom- ciplinary worlds of classical embryology, develop- ena have been implicated in the identification of mental biology, and, today, from the exciting world of etiologic and pathogenic causes of mammalian birth molecular biology.The advent of many new and excit- defects, including those of the human craniofacial ing clinical interventional strategies for the treatment regions. of birth defects now allows the clinician to treat the Normal and abnormal morphogenesis of the cran- most delicate of craniofacial abnormalities which iofacial regions, and even that of rest of the body, is were beyond the realm of treatment even for the skill- dependent upon a myriad of cell types and tissues. ful clinician due to lack of appropriate technologies. One of the most important cell types in understand- Even the details of craniofacial morphogenesis, how- ing normal and abnormal craniofacial morphogene- ever interesting to the clinician, were also seen as be- sis is the neural crest cell [2,36].While the importance ing beyond the everyday clinical realm, or esoteric, of crest cells has been hypothesized for a century or prior to the advent of the new wave clinical interven- more, not until the advent of neural crest biological tional techniques, such as high resolution imaging markers – first with isotopic labels and later with spe- and information technologies, in the fields of cranio- cific markers as monoclonal antibodies, intracellular facial and maxillofacial surgery. Today, there is a dyes, and protein assays – did the neural crest become rekindled need to understand more of the details of so widely appreciated in a variety of studies of verte- craniofacial morphogenesis, especially as that under- brate embryogenesis in general, and human craniofa- standing increases our awareness of the etiology, cial morphogenesis, in specific. While the majority of pathogenesis, and clinical features of a variety of recent neural crest studies of have necessarily dealt craniofacial defects. The goal of this chapter is to pro- with chick and mouse embryos, there is ample evi- vide a highlighted update or working understanding dence to show that basic information and associated of the “developmental blueprint” followed in human technologies gained from vertebrate embryos can be craniofacial morphogenesis, with a special focus on directly applied to neural crest cells in mammalian defects of the face, lip, palate, and associated struc- and human embryos. tures. With this in mind, prime consideration should be While recent advances in developmental and mo- given to neural crest cells because they contribute lecular craniofacial biology contribute heavily to the heavily to craniofacial morphogenesis. These impor- picture of face and palate morphogenesis, there has tant “building-block” cells arise from the final stages been an explosion in our fundamental understanding in formation of the embryonic neural tube. Neural of the very beginnings of these body regions [43]. crest cell specificity is the result of an inductive action Such new information clearly centers on the genesis, by nonneural ectoderm adjacent to the developing behavior, and developmental outcomes of many cran- neural tube (mediated by bone morphogenetic pro- iofacial “building block “ cell types throughout their teins BMP-4 and BMP-7) on the lateral cells of the life span. These fundamental phenomena include pat- neural plate as the plate transforms from a plate of ec- terns of early DNA signaling, gene and biochemical toderm into the definitive neural tube. The induced organizers,nuclear and cellular differentiation,prolif- neural crest cells express the transcription factor slug eration,migration,and,importantly,patterns of inter- which characterizes cells which separate from an em- 4 A.R. Burdi

bryonic epithelial layer and subsequently migrate as from lower cervical to the most caudal embryonic mesenchymal cells away from the parent site [33]. somites in humans. Trunk neural crest cells appear to The identification of the exact molecular mecha- have lessened migratory pathways and have fewer de- nisms and cellular events linked to the differentiation, velopmental outcomes than cranial neural crest cells, proliferation and, and especially, of the migration of including formation of spinal ganglia, sympathetic crest cells into the facial and pharyngeal regions is not ganglia, and adrenal medulla chromaffin cells. Inter- yet fully known.What is known, however, is that, with estingly, unlike cranial crest cells, trunk neural crest the variant OTX2 transcription factor, specific pat- cells do not have the capacity to differentiate into terns of neural crest proliferation and migration into skeletal tissues. the pharyngeal arches are controlled by four members The life history of cranial neural crest cells, while of homeodomain proteins called HOX genes (A-D). not of any more importance than trunk neural crest Another factor thought to be critical in the migration cells,appears to be more complex.Cranial neural crest of crest cells is a loss of cell-to-cell adhesiveness which cells are a major component of the embryo’s cephalic is associated with the loss of cell adhesion molecules end and differentiate into a wide variety of cell and (CAMs) characteristic of the neural tube and the mi- tissue types, including connective, skeletal, dentin, grating neural crest cells [91]. Following the comple- and muscle tissues of the face [64]. Unlike trunk neu- tion of craniofacial crest cell migrations and differen- ral crest cells,which show diffuse migratory pathways, tiation into specific structures (such as bones of the cranial neural crest cells follow specific migratory facial skeleton) CAMs are re-expressed. Migrating pathways into specific regions of the embryonic head. craniofacial crest cells are thought to travel through They arise from the more cephalic neural tube regions cell-free intercellular spaces and pathways that have and migrate ventrally into the pharyngeal arches ad- high levels of extracellular matrix molecules [3, 9, 10]. jacent to the upper regions of the embryonic gut tube. These migrations are determined by factors intrinsic Such migrations are extensive and follow very definite to the crest cells themselves and features of the exter- migratory paths away from the neural tube and into nal environment through which the crest cells mi- the facial and pharyngeal regions. In hindbrain re- grate. While most available information on neural gions,neural crest cells arise from eight segmented re- crest migratory pathways comes from chick studies, gions on either side of the hindbrain (rhomben- data suggest that such information is applicable to cephalon) called rhombomeres (numbered R1-R8) mammalian and humans as well. Migrations are facil- and subsequently migrate into specific pharyngeal itated by the presence of such molecular substrates as arches [12, 14]. fibronectin, laminin, and type IV collagen. Attach- Crest cells from R1 and R2 centers migrate into the ment to and migration of crest cells is mediated by a first pharyngeal arch and play important roles in the family of attachment proteins called integrins.It is im- formation of Meckel’s cartilage and the malleus and portant to note that other extracellular matrix mole- incus ear ossicles developing from it. Crest cells from cules in the pathway (e.g., chondroitin sulfate-rich R4 migrate into the second arch and contribute to the proteoglycans) can impede or block the normal mi- formation of the stapes, styloid process, and lesser gration of neural crest cells, which may lead to a num- horn of the hyoid bone.Crest cells from R6 and R7 mi- ber of craniofacial malformations. grate into the third arch, and those from R8 migrate As will be expanded upon later in this chapter,neu- into the fourth and sixth pharyngeal arches. The one ral crest cells have the remarkable capacity to differ- variant noted above is that the first pharyngeal arch entiate into a wide variety of anatomical structures also includes crest cells from midbrain levels which throughout the body. Exactly what controls crest dif- express OTX2 transcription factors. Little evidence ferentiation is still an open question. One hypothesis exists to show that crest cells from rhombomeric cen- is that all neural crest cells are equal in their develop- ters R3 and R5 play any significant role in human mental potential, and their ultimate differentiation is craniofacial morphogenesis. Crest cells initially ex- entirely predetermined by the environment through press the HOX genes from their originating rhom- which the crest cells migrate and finally reside, i.e., bomeric center, but maintenance of a specific expres- “extrinsic determinants.” A second hypothesis favors sion is dependent upon interaction of the crest cells “intrinsic determinants” and suggests that premigra- with the arch-specific mesoderm in the pharyngeal tory crest cells are intrinsically programmed for dif- arches.While there is a specific linkage between given ferent developmental fates. Recent studies indicate HOX genes and pharyngeal arches, morphologic de- that both hypotheses may be operative [1].While neu- rivatives arising from these linkages are also depend- ral crest cells have a common site of origin during ent upon epithelial-mesenchymal interactions and in- neural tube formation,not all neural crest cells behave cludes molecular signaling from surface ectoderm alike. There are two families of crest cells, i.e., cranial covering the arches,specifically,fibroblast growth fac- and trunk neural crest.Trunk neural crest cells extend tors (FGFs) which interact with underlying mes- Chapter 1 Developmental Biology and Morphogenesis 5 enchymal cells. Crest cells alone do not establish or chemical signaling from cells lining the extracellular maintain a specific pattern of morphologic expres- cleavage planes through which the crest cells migrate sion. Several regulatory factors have been identified. [37, 42]. Crest cells from the developing midbrain re- Sonic hedgehog (Shh) genes and some retinoids have gions migrate into upper facial regions, whereas crest been shown to regulate normal HOX gene expression cells from hindbrain migrate selectively into the low- within the pharyngeal arches associated with a vari- er facial regions [45–47]. Importantly, once the crest ety of developmental events, including neural plate cells migrate into specific facial regions, they differen- development and craniocaudal body pattern forma- tiate into mesenchymal cells that subsequently give tion. rise to connective tissue and muscle cells of those spe- Defective differentiation, proliferation, and migra- cific facial regions [41]. While the predominant neural tion of cranial neural crest have been linked with a va- crest-derived mesenchymal cells in the facial regions riety of developmental defects, i.e., the so-called neu- do co-mingle with mesodermally derived mesenchy- rocristopathies [24, 28, 29, 34]. Deficiencies and mal cells, the interactive nature of their co-mingling, excesses of retinoids, for example, can disrupt prolif- or lack thereof, remains uncertain. Consistent with eration and migration on specific crest cells, resulting the tenets of the “developmental field concept” [48] in in craniofacial defects, e.g., clefts of the lip and palate human morphogenesis, both human and experimen- [54, 62]. With reference to the structural abnormali- tal studies generally have hypothesized that signifi- ties in the chromosome 22-deletion DiGeorge syn- cant and early interference with normal differentia- drome, the fundamental pathogenesis for this clinical tion, proliferation, and migration embryonic cells, syndrome has been linked with defects in cranial neu- including especially the craniofacial neural crest cells, ral crest cells of the third and fourth pharyngeal arch- can lead to isolated and syndromic craniofacial de- es and cardiac outflow tract. Other cranial neuro- fects, called neurocristopathies, whose occurrence cristopathies include the wide range of craniofacial and severity depend on a combination of environ- abnormalities in the frontonasal dysplasia family, mental and genotypic factors specific to a given dys- Treacher Collins syndrome (mandibulofacial dysos- morphic or phenotypic trait [46, 47, 52]. tosis), the Robin sequence, Waardenburg syndrome Having addressed the general developmental fea- (types I and III), and neurofibromatosis (von Reck- tures of the craniofacial “building block” neural crest linghausen’s disease). cells, let’s turn our specific attention now to the key The “building block “ cells for the head and face are events in the shaping of the human face, lip, and identifiable both premorphologically and morpho- palate. When the embryo’s cephalo-caudal axis is es- logically as early as the second intrauterine week. tablished at about 14 postconception days, the facial Once mapped out these cells continue with their peak developmental field is one of the first of the head re- period of cell differentiation, proliferation, and mi- gions to appear [48]. Centrally-located in this region gration through the second intrauterine month.While is a discrete bilaminar tissue plate, called the oropha- the classical picture of craniofacial morphogenesis ryngeal membrane, whose structure and location can be framed upon the morphogenesis of primary marks the junction between the oral ectoderm and germ layer cells (i.e., ectoderm, mesoderm, endo- the endodermal digestive tube. This membrane pro- derm),there is little doubt whatsoever that the current gressively degenerates through the normal process of understandings and excitement about mammalian, apoptosis or “programmed cell death” which involves including human craniofacial, morphogenesis have increased phagocytic or lysosomal activity along the been significantly advanced by a plethora of studies of inner and outer surfaces of the membrane. Once the the origins and behavior of embryonic neural crest apoptosis of the oropharyngeal membrane is com- cells. Morphogenesis of the facial regions depends pleted at 4 weeks, there is direct continuity between heavily on the timely differentiation, directed migra- the spaces of the early oral cavity and the pharyngeal tion, selective proliferation of these crest cells which regions of the digestive tube. Only rarely does the arise as a product of neural tube formation as the neu- oropharyngeal membrane fail to degenerate. Interest- ral tube progressively pinches off from the overlying ingly,a similar ecto-endodermal membrane lies at the skin along the body’s dorsal axis. As will be discussed depth of a groove which separates the first pharyngeal later,cells and tissues within each of the embryonic fa- from the second pharyngeal arch.As will be discussed cial primordia arise from neural crest cells begin their later,this membrane will have a very different and im- migration (at about 21 postconception days) into the portant developmental fate (i.e., does not undergo facial regions, as cell clusters called rhombomeres, apoptosis) than that of the oropharyngeal membrane from their sites of origin along the portions of the related to the fact that it has,unlike the oropharyngeal neural tube which form the brain. The determinants membrane, a layer of mesenchyme interposed be- of crest cell migrations have been variously hypothe- tween its ecto- and endodermal layers. sized as including intrinsic cell “targeting”factors and 6 A.R. Burdi

Clearly as much developmental “shaping” occurs which have been linked with the failure of that specif- on the laterals aspects of the young embryo’s head as ic plate to degenerate and persist normally through- in its the frontal regions.At four weeks, a series of lat- out life as the adult eardrum, or tympanum. The ele- eral surface elevations, called pharyngeal arches, be- vated margins around the first pharyngeal groove comes quite prominent on the lateral side of the head. develop through the selective proliferation of mes- In fact, the appearance of the embryo’s head region at enchymal cells beneath the skin into six separate mes- this time closely resembles the gill slit anatomy seen enchymal swellings, called auricular hillocks. These in a comparably-staged fish embryo; however, unlike auricular hillocks progressively (from both the first in the fishes,the surface gill appearance in human em- and second pharyngeal arches) enlarge, migrate, and bryos is short-lived, except as noted above, in the case consolidate through programmed cell activity and of the development of the ear drum (or tympanum). eventually give rise to the external ear, or auricle. Fail- The pharyngeal arches contribute significantly to the ure of the auricular hillocks to develop normally can formation of the face, palate, and associated struc- result in auricles of abnormal size,shape,and position tures. Most congenital malformations of the head and as seen in a variety of isolated and syndromic cranio- neck have their beginnings during the cellular trans- facial birth defects, e.g., first and second branchial formation of the pharyngeal arches into their adult arch syndrome, hemifacial microsomia, and microtia. derivatives. For example, branchial cysts and fistulae The complete absence of the auricle (anotia) is a rare can occur in those rare instances in which human event. pharyngeal (or gill) clefts fail to smooth over on the To complete this picture of the pharyngeal arches, lateral side of the neck. As mentioned earlier, cell it is important to note that cells within the arches are masses which contribute to the bulging prominence supplied by pairs of blood vessels, called aortic arch- of the arches are the neural crest cells that have mi- es,that distribute blood from the embryonic heart up- grated into the pharyngeal arches from specific brain ward through the tissue of each arch toward the brain regions, and which eventually differentiate into mes- and then down to the body [49]. As with the pharyn- enchymal cells and give rise skeletal and muscular geal l arches themselves, not all of the aortic arches structures specific to a given pharyngeal arch. persist in humans. The aortic arches of the third, The first pair of pharyngeal arches are most impor- fourth, and sixth aortic arches do persist and become tant in shaping the human face and associated struc- greatly modified throughout the embryonic period as tures and will receive most attention in this chapter. they are reconstituted as the common carotid arteries The first pharyngeal arch,often called the mandibular which supply the neck, face, and brain. Especially im- arch,develops as two elevations around the oral open- portant in this dynamic development of the craniofa- ing which was filled in earlier by the oropharyngeal cial vasculature is the shifting of the primary arterial membrane. The larger and lower regions of this arch supply to the embryonic face prior to, during, and fol- form much of the mandibular anatomy and the lowing the formation of the secondary palate. Unlike malleus and incus middle ear ossicles, whereas the in the adult, prior to the seventh week, the primary smaller and upper regions of the first arch on either source of blood to both the superficial and deep head side of the oral opening give rise to the anatomy of up- tissues is the internal carotid artery and its branches. per lip, teeth, maxilla, zygomatic bone, and squamous At about 7–8 weeks when the embryonic palatal portions of the temporal bone. The second pharyn- shelves are experiencing their most critical stages of geal arch is located beneath the first arch and is often elevation and closure, an important shift occurs in the called the hyoid arch in that it contributes significant- primary blood supply to the face and palatal tissues ly to the formation of the hyoid bone and one of the from the internal carotid to the external carotid arte- three middle ear ossicles, called the stapes. These two rial system. This transition involves a temporary vas- pharyngeal arches, like each of the other four pharyn- cular shunt between internal and external carotid sys- geal arches,are separated from each other by a surface tems provided by the stapedial artery.Failure of either pharyngeal l groove which grows inwardly to meet an the stapedial artery to form or failure of a complete endodermal-lined outpocketing from the developing and timely transition to occur has been hypothesized pharyngeal region, i.e., the first pharyngeal pouch. As in identifying the pathogenesis of such conditions as is the case with most pharyngeal grooves and pharyn- palatal clefting and mandibulofacial dysostosis [51]. geal pouches, the contact zone between a pharyngeal l Considerably dependent upon the timely set mor- groove and a pharyngeal pouch is a bilaminar plate of phogenic events that occur from the time of implanta- ectoderm and endoderm which eventually degener- tion through the fourth week,the embryonic face con- ates, again through the process of “programmed cell tinues through its “developmental critical period,” death” and increased phagocytic activity. In the case which spans the fifth through seventh intrauterine of the first arch, however, this bilaminar plate is sepa- weeks. It is that time period during which in human rated by invading crest-derived mesenchymal cells craniofacial morphogenesis generally is most suscep- Chapter 1 Developmental Biology and Morphogenesis 7 tible to either known or suspected birth defect-pro- enlarge and migrate medially toward each other and ducing agents, or teratogens [66]. Arising from the the lateral and medial nasal prominences [12]. This first pharyngeal arch are four primordial or “building migration is associated not only with patterns of cel- block” tissue masses that surround the large central lular growth within the maxillary prominences, but depression of the primitive oral cavity. Continued also with timely migration of the eye fields from the morphogenesis of the facial prominences depends lateral to the frontal regions of the embryo’s face dur- heavily upon the continuing migration, proliferation, ing the fifth through eighth weeks [8]. Disturbances in and differentiation of the neural crest cells, under the normal eye field migration have been suggested as direction of developmental morphogens, to a point in one possible cause of median facial clefting and the time when the facial prominences, or primordia, are conditions of hypo- and hypertelorisms. Continued clearly identifiable as the single median frontonasal medial migration of the maxillary prominences on prominence, paired maxillary prominences on either both sides also moves the medial nasal prominences side of the frontonasal process, and two mandibular toward the midline and each other. By the end of the prominences beneath the oral opening.The shape and sixth week, each maxillary prominence blends, or size of these prominences as well as development of merges, with the lateral nasal prominence along a line the specific skeletal and muscular structures of each which demarcates the future nasolacrimal groove and pharyngeal arch are critically dependent upon the duct. This event then establishes the continuity be- continued viability and differentiation of the neural tween the side of the nose, or alar region, formed by crest cells which are especially sensitive to teratogens, the lateral nasal prominence with the cheek region e.g., cortisone and retinoic acid [50, 61]. formed from the maxillary prominence. A combina- Continuing further with our focus on the develop- tion of reduced cell numbers and abnormal migration mental “blueprint”for the face, specifically the lip, it is of mesenchymal cells can lead to the abnormal merg- important to note that the outcomes of several dis- ing or consolidation of the maxillary and lateral nasal tinct brain-skin interactions in placode formation are prominences. Although seen infrequently, this can also essential in early facial morphogenesis.By the be- lead to facial defects involving oblique facial clefts, ginning of the fifth week, oval patches of skin ecto- persistent nasolacrimal grooves, and failure of the na- derm lateral to the median frontonasal prominence solacrimal duct to develop. interact with brain tissue to set off an ecto-ectodermal Between the fourth and eight weeks, the medial interaction resulting in the development of the two nasal prominences merge with each other, small low- thickened nasal placodes located at the ventrolateral er portions of the lateral nasal prominences, and with regions of the frontonasal prominence. Neural crest- cells in the larger maxillary prominences. This sub- derived mesenchymal cells along the margins of the surface merging of cells, especially between the medi- nasal placodes proliferate rapidly to produce horse- al nasal and maxillary prominences, results in the shoe shaped elevations around the placode, called the continuity of upper jaw and lip.As part of this consol- medial and lateral nasal prominences, whose contin- idation of the medial nasal and maxillary promi- ued rapid growth gradually forms the nasal pits, or nences in upper lip formation, two important mor- early nostrils. The forward growth of each lateral phologic events need to occur. First, there is a nasal process forms the ala of the nose, whereas the deepening and downward growth of the nasal pit to- medial nasal process contributes to the formation of ward the oronasal cavity as a blind-ending sac whose the nose tip, columella, the philtrum, tuberculum, and floor eventually degenerates through programmed- frenulum of the upper lip, and the entire primary cell death resulting in the formation of the primitive palate. Through the process of relative growth in this choanae, which allows a continuity between the area, the nasal placodes gradually “sink” to the depth spaces of the primitive nasal cavity and the common of each nasal pit. Failure of the nose to develop com- oronasal cavity. An event occurring concomitantly pletely is associated with failure of both nasal pla- with nasal pit morphogenesis is the formation of the codes to develop. A second important skin-brain in- seam between the intermaxillary segment and the teraction gives rise to localized thickenings of surface maxillary prominence. As these two segments come ectoderm on each side of the embryo’s head which together in the sixth week, the developmental surface will form the optic lens, retina, and nerve. Important- seam of cells between them also elongates as the nasal ly, and as will be discussed later, these eye fields are pit elongates, deepens, and moves downward. This de- first located on the lateral aspects of the embryo’s velopmental seam, called the nasal fin, essentially head and progressively migrate to the frontal midline forms the floor of the nasal pit and progressively de- at about the time the facial prominences are consoli- generates by increased activity among phagocytic dating into the complete face [8]. cells on either side of the seam [67]. Once “pro- Selective differentiation and proliferation of mes- grammed cell” death of the nasal fin is essentially enchymal cells cause the maxillary prominences to completed at about the seventh week, mesenchymal 8 A.R. Burdi

cells from both the intermaxillary and maxillary and consolidation of the five major facial promi- prominences intermix, leading to fusion of the upper nences, a recognizable human face is evident by the lip segments into the upper lip and its cupid’s bow. end of the eighth prenatal week [11, 40, 55, 58, 59]. The completion of the embryonic lips generally oc- Morphogenesis of the mammalian palate is an even curs about 1 week earlier that the formation of the more complex process which depends heavily upon palate. Thus, the lips and palate have different “devel- the balance of genetic, hormonal, and various growth opmental critical periods” and, as such teratogens factors. As the face nears the completion of its “devel- might affect either the lips or palate separately, or in opmental critical period,” lateral palatine processes combination. The intermixture of mesenchymal cells which form the secondary palate grow out from the within the consolidated lip segments give rise to con- walls of the still common oronasal cavity. The “devel- nective tissue components and muscle fibers within opmental critical period” for the palate is from the the orbicularis oris ring of the upper lip. Complete or end of the sixth week through the eighth intrauterine incomplete failure of the nasal fin to degenerate have week, or 1 week longer in duration than that of the lip. been associated with unilateral and bilateral clefts of These palatine shelves first grow medially, then be- the upper lip which variously involve abnormalities of come oriented inferolaterally to lie on either side of the orbicularis oris muscle in terms of the numbers the tongue, which is quite precocious in its own devel- and distribution of its muscle fibers as part of the or- opment as a muscle-filled epithelial sac that fills much bicularis ring. of the oronasal cavity. Nearing 8 weeks, the vertically The incidence of orofacial clefts varies in accor- oriented palatine shelves are progressively reposi- dance with the variances reported for differing popu- tioned above the tongue mass. This repositioning of lation groups [22]. Examples of such population-spe- the shelves is thought to involve a combination of con- cific differences, called polymorphisms, include cleft current events, including a downward contraction of lip with or without cleft palate, which is one of the the tongue, an ameboid-like reshaping of the shelves most common craniofacial birth defects in human which gradually places them over the tongue surface, with a reported incidence as high as 1:1000 in whites an increase in extracellular shelf “forces” (or shelf flu- and higher in Asian and lower in black populations id turgor) which reposition the shelves in a horizontal [13, 38, 56]. Another example of population polymor- position, and a downward repositioning of the lower phisms shows that isolated cleft palate occurs more jaw [7,18,19].In reality,normal or abnormal horizon- often in females (67%) than in age-matched males. talization of the palatine shelves is related to a combi- This gender difference has been associated with a nation of these three events. Palatal shelf elevation be- longer period of time for palatal closure in females gins in the posterior regions of the shelves and which essentially increases the time during which fe- depresses the tongue downward and forward and this male embryos might be affected by palatogenic ter- allows the more anterior regions of the shelves to first atogens [6]. Lateral clefts of the lip may or may not be contact one another near the posterior edge of the pri- associated with clefts of the palate. Mesenchymal cell mary palate, or in the region of the future incisive deficiency that results in partial or complete failure of canal [4]. Once the shelves are in a horizontal posi- the two medial nasal prominences to consolidate into tion, the shelves contact each other, and essentially a philtrum can contribute to the formation of such de- stick together by a combination of interlocking shelf fects as a bifid nose, or the rare median cleft (“hare surface microvilli and a proteoglycans surface coating lip”) of the upper lip, as characteristically seen in the along the medial epithelial edge (MEE) of each shelf autosomally recessive Mohr syndrome [23, 25, 30]. [39]. The elimination of the MEE is crucial for normal As the consolidation of facial processes progresses morphogenesis of the anterior regions of the second- through the embryonic period, crest-derived mes- ary palate [15, 20, 26, 32]. Once the shelves make con- enchymal cells within the maxillary prominences rap- tact, there is a degeneration (i.e., apoptosis or pro- idly proliferate and differentiate into tissues which grammed cell death) of epithelial cells along the form mesenchymal cell fields from which the muscles abutting shelf linings, and a directed movement of of facial expression develop, and whose myofibers are crest-derived mesenchymal cells from one shelf to the innervated by the cranial nerve to the second arch, other. This process of epithelial degeneration along i.e., the facial nerve. Similarly, crest-derived mes- with intershelf bridging of mesenchymal cells is enchyme in the maxillary and mandibular portions of called fusion. the first pharyngeal arch differentiate predominately As is the case for craniofacial morphogenesis in into the muscles of mastication, which are innervated general, several categories of factors (e.g., chromo- by the trigeminal nerve of the first pharyngeal arch. somes, genes, signaling proteins, transcription fac- Cells within the mandibular prominence give rise to tors, specific proteins) have either been identified or muscle and connective tissue structures of the lower hypothesized as important in normal and abnormal lip, chin, and lower cheek regions. With the reshaping palatogenesis [57, 64]. Fusion of the palatal shelves Chapter 1 Developmental Biology and Morphogenesis 9 has been linked to a variety of growth factors like etiology of clefts of the human lip and palate, are still those in the TGF-Beta-3 growth factor family [32, 53, wanting. Crucial faulty chromosomes (see, for exam- 63] and protein activities [16,31].Complete or region- ple,ref.17) and genes linked to inherited forms of cleft al failures in programmed cell death MEE along the lip and palate have recently been identified. This can- lengths of the palatal shelves can lead to various forms didate gene is known as Interferon Regulatory Factor of palatal clefts.While great strides have been made in 6 [IRF6] [35]. Other candidate genes have been as- identifying genetic,cellular,and molecular controls of signed some importance in palatal morphogenesis, normal palatal development (mostly in mice and including MSX1, LHX8 6p24 genes (associated with chick embryos),the identification of the exact balance palatal shelf growth and differentiation); TGFA, EGFR between intrinsic and environmental controls of and HOXA2 (associated with elevation and depres- palatal morphogenesis remains elusive [44, 60]. The sion of tongue);TGFB3 and PVRL1 (associated with embryonic palatine raphe, or future midpalatine su- associated with sequential fusion stages along the ture, marks the line of fusion between the palatine midpalatal seam), and TGFA and EGFR (associated shelves. From the site of first shelf contact and fusion with actual apoptosis, or “programmed cell death” near the future incisive foramen, fusion of the more events along the midpalatal seam). posterior regions of the shelves takes place over the Some clefts of the lip with or without cleft palate next two weeks. Fusion also occurs between the are seen regularly in a number of single mutant gene shelves and the inferior edge of the nasal septum, ex- syndromes. Other clefts are associated with chromo- cept in the more posterior regions where the soft somal syndromes, especially in trisomy 13. A com- palate and uvula remain free. Once fusion of the plete cleft palate represents a maximum degree of shelves of the secondary palate is complete, their mes- clefting and is a birth defect in which the cleft extends enchymal cells differentiate into osteogenic cells from the incisive foramen region through the soft which form the skeletal elements of the premaxillary, palate and uvula. The incisive foramen region is the maxillary, and palatine portions of the palate. demarcation used in distinguishing the two major Formation of the soft palate and uvula takes a groups of cleft lip and palate. Anterior cleft types in- slightly different course than that of the regions of the clude cleft lip,with or without a cleft of the alveolar re- secondary palate which give rise to the hard palate [5]. gion of the maxilla. A complete anterior cleft extends The soft palate and uvula develop from two separate through the lip and alveolar region to the incisive masses found at the most posterior portions of the foramen region. The pathogenesis of anterior clefts is secondary palatine shelves. Unlike the fusion mecha- related to a deficiency of neural crest-derived mes- nism which is in place along much of the length of the enchymal cells chiefly within the intermaxillary seg- palatine shelves, the consolidation of these two sepa- ment of the lip. The posterior cleft type of birth defect rate masses is brought about by a selective prolifera- generally include clefts of the secondary palate that tion of mesenchymal cells located deep in the valley extend from the incisive foramen through the soft between the masses. As that proliferation, called palate and uvula. The observation that the female sec- merging, continues the valley between the two distal ondary palate has a longer “developmental critical pe- shelf masses is obliterated, which results in a riod” [6] than the male embryo by approximately smoothening of the contour of the soft palate and 1 week offers some explanation why isolated cleft uvula. Failure of the merging process in soft palate palate is more prevalent in females (66%) than males and uvula development can result in complete or par- (34%). In general, the pathogenesis of posterior tial clefts of the soft palate and uvula. palatal clefts is related to abnormalities in a combina- Clefts of the palate, with or without clefts of the lip, tion of events ranging from deficiencies in mesenchy- are relatively common depending on the population mal cell numbers to perturbations in the shelf extra- group of the individual [23, 65]. Whereas occurrence cellular matrices to abnormal elevation and fusion of figures for nonsyndromic cleft lips (with or without the shelves, or lack thereof, as associated with a num- cleft palate) are about 1 in 1,000 live births, clefts of ber of hypothesized teratogens, including excess dos- the palate (with or without cleft lip) occur in 1 in 2,500 es of retinoic acids, glucocorticoids, and dioxins. live births, again depending on the population group of the individual, i.e., highest incidence in Asian, in- termediate in white, and lowest in black populations 1.1 Summary [60]. Most clefts of the lip and palate generally are re- lated to an interplay of genetic and environmental fac- The understanding of the natural history, clinical de- tors, i.e., multifactorial inheritance [21].While animal lineation, and clinical management of birth defects studies have provided some insight into the molecular involving the face, lip, and palate has progressed sig- and cellular bases of these defects, precise etiologic nificantly over the last 20 years and continues to do as explanations, especially involving teratogens in the we move further into the 21st century. Although hu- 10 A.R. Burdi

man craniofacial morphogenesis is clearly the culmi- abnormal. References noted with an (*) are texts con- nation of a very complex series of diverse and over- taining illustrations that elucidate the information lapping developmental events, all of these events can contained in this chapter. be categorized into four fundamental processes which span mammalian development and are evident in the earliest beginnings of the face and palate – normal References and abnormal: (1) cell differentiation – the process through which the myriad of “building block” cell 1. Anderson DJ. Cellular and molecular biology of neural types invoked in facial morphogenesis are generated crest lineage determination. Trends Genet 1997; 13:276– from the single-celled zygote; (2) morphogenesis – the 280. 2. Birgbauer E, Sechrist J, Bronner-Fraser M, Fraser S. Rhom- process or set of processes through which the complex bomeric origin and rostro caudal reassortment of neural form of the face and its constituent cells, tissues, and crest cells revealed by intravital microscopy. Development organs will emerge in a timely fashion along pattern- 1995; 935–945. able individual and population lines; (3) growth – the 3. Brinkley L, Morris-Wiman J. The role of extracellular ma- collective results of differentiation and morphogene- trices in palatal shelf closure. In: Zimmerman EF (ed) Cur- sis; and (4) dysmorphogenesis and abnormal growth – rent trends in developmental biology. New York: Academic this is the most exciting of the challenges we face to- Press; 1984. 4. Burdi AR, Faist K. Morphogenesis of the palate in normal day as we strive to understand how environmental in- human embryos with special emphasis on the mechanisms fluences interact with and cause changes in the ex- involved. Am J Anatomy 1967; 120:149–160. pression of the genetic factors governing the behavior 5. Burdi AR. Distribution of midpalatine cysts: A re-evalua- of those cells which will give rise to the entire human tion of human palatal closure mechanism. J Oral Surg 1968; body, and especially the face and palatal regions. The 26:41–45. treatment of defective genes is very much a part of the 6. Burdi AR, Silvey R. Sexual differences in closure of the hu- current clinical agenda dealing with craniofacial de- man palatal shelves. Cleft Palate J 1969. 6:1–7. 7. Burdi AR, Feingold M, Larsson KS, Leck I, Zimmerman E. fects. The basic scientist, the dysmorphologist, the cli- Etiology and pathogenesis of congenital cleft lip and cleft nician, and, importantly, those with natural or ac- palate. An NIDR State of the Art Report. Teratology 1972. quired craniofacial defects have gained significant 6:255–270. advantage from the critical use of available informa- 8. Burdi AR, Lawton TJ, Grosslight J. Prenatal pattern emer- tion coming from classical and experimental studies gence in early human facial development. Cleft Palate J of human morphogenesis. These advantages will con- 1988; 25:8–15. tinue to increase as laboratory scientists and clinician 9. Bronner-Fraser M. Experimental analyses of the migration and cell lineage of Avian neural crest cells. Cleft Palate J scholars move rapidly together into the world of mo- 1990; 27:110–120. lecular and gene biology. These approaches should 10. Bronner-Fraser M, Fraser SE. Cell lineage analysis of the and will increase our knowledge base on the patterns avian neural crest. Development 1991; 2:17–22. and underlying causes of normal and abnormal cran- 11.* Carlson BM.Human embryology & developmental biology. iofacial morphogenesis – and our patients will be all 2nd.ed. St. Louis: Mosby; 1999. the better for it. However, most researchers and treat- 12. Carstens MH. Development of the facial midline. J Cranio- ment providers well realize that the practical transfer facial Surg 2002; 13:129–187. 13. CDC National Center on Birth Defects and Developmental of new biological information on normal and abnor- Disabilities. 2002. http://www.cdc.gov/ncbdd. mal development flowing from the laboratory bench 14. Couly G,LeDourain NM.Head morphogenesis in embryon- to the clinical bedside may be neither easy nor timely ic avian chimeras: evidence for a segmental pattern in the to achieve in the effective treatment and management ectoderm corresponding to neuromeres. Development of craniofacial abnormalities. 1990; 108:543–558. Epilogue. “And the end of all of our exploring will 15. Cuervo R, Valencia C, Chandraratna RAS, Covarrubias L. be to arrive where we started and know the place for Programmed cell death is required for palatal shelf fusion and is regulated by retinoic acid. Dev Biol 2002; 245:145– the first time.” T.S. Eliot, 1918 156. 16. Darling DS, Stearman RP, Qiu M, Feller JP. Expression of Zfhep/EF1 protein in palate, neural progenitors, and differ- References ential neurons. Gene Expr Patterns 2003; 3:709–717. 17. Davies AF. Further evidence for the involvement of human The following references have been selected from an chromosome 6p24 in the aetiology of orofacial clefting. J. ever-growing literature in developmental biology and Med Genetics 1998; 35:857–861. 18. Diewert VM. The role of craniofacial growth in palatal shelf craniofacial biology in particular. Perusal of any one elevation. In: Pratt RM, Christiansen RL (eds). Current re- or several of these references should lead the reader to search trends in prenatal craniofacial development. New further readings which bear on the subject of the biol- York: Elsevier North-Holland; 1980. ogy of face, lip and palate development – normal and Chapter 1 Developmental Biology and Morphogenesis 11

19. Ferguson MWJ. Palatal development. Development 1988; 40. Moore K, Persaud TVN. The developing human. 6th ed. 103:41–57. Philadelphia: W.B. Saunders; 1998. 20. Fitchett JE, Hay ED. Medial edge epithelium transforms to 41. Morris-Kay G, Tan S-S.Mapping neural crest cell migration mesenchyme after palatal shelves fuse. Dev Biol 1989; 131: pathways in mammalian embryos. Trends Genet 1987; 455–474. 3:257–261. 21. Fraser FC. The multifactorial threshold concept – uses and 42. Morris-Kay G, Tuckett F. Early events in mammalian cran- abuses. Teratology 1976 14:267–280. iofacial morphogenesis. J Craniofac Genet Dev Biol 1991; 22. Freni SC, Zapisek WF. Biologic basis for a risk assessment 11:181–191. model for cleft palate. Cleft Palate J 1991; 28:338–346. 43. Nieto MA. Control of cell behavior during vertebrate devel- 23. Gelehrter TD, Collins FS, Ginsberg D. Principles of medical opment by Slug, a zinc finger gene. Science 1994; 264:835– genetics. 2nd ed. Baltimore: Williams & Wilkins; 1998. 839. 24. Goodman RM, Gorlin RJ. The malformed infant and child. 44. Murray J. Gene/environmental causes of cleft lip and/or New York: Oxford University Press; 1983. palate. Clin Genet 2002; 61:248–256. 25. Gorlin RJ, Cohen MM Jr., Levin LS. (eds) Syndromes of the 45. Noden DM. Origins and patterning of craniofacial mes- head and neck 4th ed. New York: Oxford University Press; enchymal tissues. J Craniofac Genet Dev Biol 1986; 2:15–31. 2002. 46. Noden DM.Cell movements and control of patterned tissue 26. Griffith CM, Hay ED. Epithelial-mesenchymal transforma- assembly during craniofacial development. J Craniofac tion during palatal fusion: carboxyfluorescein traces cells Genet Dev Biol 1991; 11:192-213. at light and electronmicroscopic levels. Development 1992; 47. Noden DM. Vertebrate craniofacial development: the rela- 116:1087–1099. tion between ontogenetic process and morphologic out- 27. Hunt P,Wilkinson D, Krumlauf R. Patterning the vertebrate come. Brain Behav Evol 1991; 190–225. head: murine Hox 2 genes mark distinct subpopulations of 48. Opitz JM. The developmental field concept New York: Liss; premigratory and migratory cranial neural crest. Develop- 1986. ment 1991; 112:43–50. 49. Padget D.The development of the cranial arteries in the hu- 28. Johnston MC, Bronsky PT. Craniofacial embryogenesis: man embryo. Carnegie Contrib Embryol 1948; 32:205–268. abnormal developmental mechanisms. Ch. 5:61–124. In: 50. Persaud TVN. Environmental causes of human birth de- Mooney MP, Siegel ML, (eds) Understanding craniofacial fects Springfield: Charles C Thomas; 1990. anomalies. New York: Wiley and Liss; 2002. 51. Poswillo DE. The pathogenesis of the first and second 29. Jones KL. Smith’s recognizable patterns of human malfor- branchial arch syndrome. Oral Surg, Oral Med, Oral Pathol mation. 4th ed. Philadelphia: W.B. Saunders; 1990. 1973; 35:302–328. 30. Jones M. The neurocristopathies: reinterpretation based on 52. Pratt RM. Environmental factors influencing craniofacial the mechanism of abnormal morphogenesis. Cleft Palate J morphogenesis. In: Vig KWL, Burdi AR (eds). Craniofacial 1990; 27:136–149. morphogenesis and dysmorphogenesis. Craniofacial growth 31. Kang P, Svoboda KKH. PI-3 kinase activity is required for series. Monograph 21. Ann Arbor: University of Michigan epithelial-mesenchymal transformation during palatal Center for Human Growth & Development; 1988. fusion. Dev Dynamics 2002; 225:316–321. 53. Proetzel G,Pawlowski SA,Wiles MV,Yin M,Boivin GP,How- 32. Kaartinen V,Cui XM, Heisterkamp N, Groffen J, Shuler CF. els PN, Ding J, Ferguson MW,Doetschman T. Transforming Transforming growth factor-beta 3 regulates transdifferen- growth factor-beta 3 is required for secondary palate fu- tiation of medial edge epithelium during palatal fusion and sion. Nat Genet 1995; 11:409–414. associated degradation of the basement membrane. Dev 54. Rowe A, Richman JM, Brickell PM. Retinoic acid treatment Dynamics 1997; 209:255–260. alters the distribution of retinoic acid receptors in the em- 33. Keynes R, Cook GMW.Axon guidance molecules. Cell 1995; bryonic chick face. Development 1991; 111:1007–1016. 83:161–169. 55. Sadler TW. Langman’s medical embryology. 9th ed. Balti- 34. Kissel P,Andre JM, Jacquier A. The neurocristopathies New more: Lippincott Williams & Wilkins; 2004. York: Masson; 1981. 56. Schutte BC, Murray J. The many faces and factors of oro- 35. Kondo S, Schutte BC, Richardson RJ, Bjork BC, Knight AS, facial clefts. Hum Mol Genet 1999; 8:1853–1859. Watanabe Y,Howard E,Ferreira de Lima RLL,Daack-Hirsch 57. Slavkin H. Regulatory issues during early craniofacial S, Sander A, McDonald-McGinn DM, Zackai EH, Lammer development. Cleft Palate J 1990; 27:101–109. EJ, Aylsworth AS, Ardinger HH, Lidral AC, Pober BR, 58. Sperber GH. Craniofacial embryology. 5th ed. Hamilton, Moreno L, Arcos-Burgos M, Valencia C, Houdayer C, Canada: BC Decker; 2001. Bahuau M, Moretti-Ferreira D, Richieri-Costa A, Dixon MJ, 59. Sperber GH.First year of life: prenatal craniofacial develop- Murray JC. Mutations in interferon regulatory factor 6 ment. Cleft Palate J 1990; 29:109–111. cause vander Woude and popliteal pterygium syndromes. 60. Spritz RA. The genetics and epigenetics of orofacial clefts. Nat Genet 2002; 32:285–289. Curr Opin Pediatr 2001; 13:556–560. 36. Le Dourain N. The neural crest. Cambridge, England: Cam- 61. Sulik KK, Dehart DB. Retinoic-acid induced limb malfor- bridge University Press; 1982. mations resulting from apical ectodermal ridge cell death. 37. Lumsden A, Sprawson N, Graham A. Segmental origin and Teratology 1988; 37:527–537. migration of neural crest in the hindbrain region of the 62. Sulik KK, Johnston MC, Cook CS, Webster WS. Teratogen- chick embryo. Development 1991; 113:1281–1291. sand craniofacial malformations. Dev Biol 1988; 103:213– 38. March of Dimes Birth Defects Foundation. 2002. 232 http://www.modimes.org/. 63. Taya Y,O’Kane S,Ferguson MW.Pathogenesis of cleft palate 39. Martinez-Alvarez C, Tudela C, Perez-Miguelsanz J, O’Kane in TGF-beta 3 knockout mice. Development 1999; 126: S, Puerta J, Ferguson MW. Medial edge epithelium cell fate 3869–3879. during palatal fusion. Dev Biol 2000; 220:343–357. 12 A.R. Burdi

64. Thesleff I, Nieminen P. Molecular mechanisms of cell and 66. Vig KWL, Burdi AR. Craniofacial morphogenesis and dys- tissue interactions during early tooth development. Anat morphogenesis. Craniofacial Growth Series Monograph Rec 1996; 245:151–161. No.21. Ann Arbor; The University of Michigan Center for 65. Thompson MW, McInnes RR, Willard HF. Thompson & Human Growth & Development; 1988. Thompson genetics in medicine. 5th ed. Philadelphia: W.B. 67. Warbrick JG. The early development of the nasal cavity and Saunders; 1991. upper lip in the human embryo. J Anat 1960; 94:351–362.