The mélange of molecular mechanisms involved in the cascading events of embryogenesis are predicated by the expression patterns of specific genes contained in the human genome, constrained by impacting environmental factors. Gene expression patterns are revealing regions of the emerging embryo that have been previously observed histologically and anatomically but not heretofore realized as genetically distinct entities during development. Studies of animal model systems have contributed to our understanding of the molecular determinants that contribute to facial patterning (Swartz et al. 2011). Herein is the marriage of genetics with developmental biology becoming of potential clinical significance.

Current investigations are delineating the complex molecular embryology of development. Specific defects in molecular pathways and networks may provide insights into the etiology of clefting. While embryologists focus on the mechanisms of malformation, deformation, disruption, and dysplasia, clinicians focus on the etiology, diagnosis, treatment, prognosis, and prevention of clefting. The incidence and epidemiology of orofacial clefts provide some clues to the etiology of these malformations (Mossey 2007). The combination of basic and clinical sciences should provide the ideal goal of comprehensive cleft care.

In establishing etiology, it would be useful to have available the gene expression patterns and flow of biochemical pathways underlying morphogenic events. Understanding the local regulation of cellular behaviors and misregulation of any step in these processes can provide insights into clefting consequences. The recognition of the molecular and tissue elements responsible for normal labiopalatogenesis will allow prognostication of clefting defects in their deficiencies. The therapeutic application of growth factors and gene therapy has the potential for biomimetic preventive and healing regimens (Scheller and Krebsbach 2009). Scarless repair of clefts is now potentially feasible (Larson et al. 2010).

Of the estimated 25,000 protein-coding genes in the human genome, some 17,000 genes have been identified in contributing to craniofacial development (International Human Genome Sequencing Consortium 2004). Engineering advancements in genome sequencing technology with the advent of next-generation massively parallel sequencing platforms has made possible the 1,000 Genomes Project which is cataloguing human genotypic variations in all of these genes (Nielsen 2010). The application of whole exome sequencing has proven to be a powerful tool in its ability to identify genes that influence craniofacial patterning (Ng et al. 2010), with whole genome sequencing being the goal of personal medicine for genetic risk assessment and prevention.

The complexity of contributions of the hundreds of genes to facial formation is being elucidated by identifying each gene’s individual expression for each stage of development (Feng et al. 2009). Identification of gene mutations responsible for craniofacial syndromes provides clues to the genetic basis for craniofacial clefting. However, detailed molecular and cellular analyses of the chronology and loci of gene expression patterns, upon which are superimposed epigenetic phenomena, make unraveling the complexity of craniofacial morphogenesis a daunting challenge. The ever-constant new identification of gene expression profiles of embryonic craniofacial and oral structures has led to the development of a consortium titled COGENE (Craniofacial and Oral Gene Expression Network) that can be accessed online at Cogene: http://hg.wustl.edu/COGENE/ (Cai et al. 2005). Therein is contained a list of all hitherto identified genes, growth factors, and signals involved in the expression profiles of structures between the 4th and 8.5 weeks of human development. It is in the mutation or silencing of genes or the misappropriation of growth factors and signals that the source of some developmental defects is revealed. The intricacies of RNA editing, complex regulatory networks, crisscrossing molecular pathways, together with overlapping and redundancies of gene expression patterns make unravelling the skein of individual influences particularly difficult. A FaceBase consortium providing a comprehensive program of craniofacial research has been established (Hochheiser et al. 2011).

Some of the genes implicated in craniofacial development through human and animal model studies are listed in Table 1.1.

Ascribing specific functions to all these genes is the aim of molecular biology, but it is the realization of the biology encoded within each gene that will provide comprehension of developmental phenomena and their aberrations. Genomic analyses are revealing the molecular architecture behind complex developmental pathways (Brito et al. 2011; Swartz et al. 2011), and the multifactorial basis of the etiology of clefts (Abu-Hussein 2012).

Human genetics can lead to insights of phenotypic diagnosis and provide understanding of the relationships of components of molecular circuitry that will improve the ability of genotypic information to predict the phenotype of complex clefting traits (Dixon et al. 2011; Rahimov et al. 2011; Stuppia et al. 2011; Yuan et al. 2011).

The majority of orofacial clefting cases are nonsyndromic and have no identified cause. Genetic and phenotypic heterogeneity of facial clefting has complicated the identification of the responsible phenomena. Research studies have identified genetic variants associated with cleft lip and palate through linkage analysis, candidate gene approaches, direct sequencing, deletion and duplication analysis by array comparative genomic hybridization (aCGH), and genome-wide association studies (GWAS) (Rahimov et al. 2011; Weatherley-White et al. 2011). A genome-wide association study has identified five genetic loci influencing facial morphology in Europeans: PRDM16, PAX3, TP63, C5o rf 50 and COL17A1 (Liu et al. 2012). This study established links between DNA variants previously associated with non-syndromic cleft lip/palate at 2p21, 8q24, 13q31 and 17q22. The complexity of clefting is illustrated in the numerous different types of genes associated with the phenotype. Such studies have led to the characterization of several genes with variants that convey an increased risk of orofacial clefting including MSX1, a transcription factor expressed in the anterior palate (Jezewski et al. 2003; van den Boogaard et al. 2000), and TGFβ3, a signaling cue involved in cell migration and palatal shelf fusion (Ashique et al. 2002; Iordanskaia and Nawshad 2011; Lidral et al. 1998). TBX22, which functions in conjunction with SUMO1 (Shi et al. 2009) as a transcriptional repressor, participates in posterior palate osteogenesis (Andreou et al. 2007) and whose mutations cause X-linked cleft palate and ankyloglossia (Kantaputra et al. 2011). Another transcription factor is SATB2, which contributes to osteoblastogenesis and influences expression of transcription factors Alx4, Pax9, and Msx1 in the base of the developing palates of mouse models (Britanova et al. 2006; FitzPatrick et al. 2003; Zhang et al. 2011b). The genetic variants contributing to the highest incidence of CL/P (∼2 % of all cases) are found in the interferon regulatory factor 6 (IRF6) gene, a transcription factor that participates in the differentiation of keratinocytes in the developing epidermis (Ingraham et al. 2006; Kondo et al. 2002; Richardson et al. 2006). Mutations or microdeletions in IRF6 are responsible for Van der Woude and popliteal pterygium syndromes, both of which exhibit variable phenotypic expression of labiopalatal clefting and lip pit depressions (Jobling et al. 2011; Kondo et al. 2002). A genome-wide meta-analysis of non-syndromic cleft lip with or without cleft palate has identified six new susceptibility regions viz. 1p36, 2p21, 3p11.1, 8q21.3, 13q31.1 and 15q22 (Ludwig et al. 2012). Variants of SKI have been proposed as a candidate gene for non-syndromic clefts of the lip and palate (Mangold et al. 2012).

Genetically determined elements such as facial and nasal width, bizygomatic distances (Boehringer et al. 2011), palatal height, and jaw growth constitute an additive to the genotype that approaches a cleft palate threshold, whereby each element contributes only a small increase in risk. This threshold may be crossed by external factors that include environmental influences, such as smoking, and potential epidemiological factors including maternal stress and age (Fraser 1976; Jagomagi et al. 2010; Wallace et al. 2011; Wehby et al. 2011; Zhang et al. 2011a; Wu et al. 2012). Maternal smoking during pregnancy implicates TGFB3 and MN1 in the etiology of submucous cleft palate (Reiter et al. 2012).

The functional role of many of these genes has yet to be fully established. Before a link between a gene and its expressed phenotype is recognized, there needs to be extensive characterization of the gene’s products. The topographical areas and timing of gene expression need to be known for specific regions of orofacial development. Interruption of components of the genetic-metabolic machinery responsible for normal embryonic development can lead to malformations. Disharmonic growth between embryonic components occasioned by subtle differences in the number of cell divisions or in the onset or offset times or rates of cellular activities may variably contribute to dysplasias. The biochemical basis of development and growth changes with time during different stages of development.

In a clinically oriented text, consideration of the very early stages of embryogenesis involving molecular biological mechanisms, induction by signaling factors, tissue differentiation, histogenesis, and organ morphogenesis and growth, each of which constitute enormous fields of study, must be greatly condensed. Appreciation of these underlying developmental phenomena is necessary in understanding the series of cascading events leading from the initial zygote formed by the union of parental gametes to the fully fledged infant (Fig. 1.3). Aberrations or variations from the normal morphogenetic patterns, whether of genetic, epistatic, or environmental origin, are responsible for many of the congenital anomalies that constitute clinical syndromes. Currently, there are no specific tests available for genetic susceptibility to orofacial clefts. The revelation of associated congenital anomalies with cleft lip and palate may further identify the interrelationships of diverse embryonic developments with genetic mutations held in common.

The relevance of embryological understanding of facial development is becoming increasingly significant not only for seeking the etiology of orofacial anomalies but also for the application of the molecular mechanisms of normal embryogenesis to the emerging fields of genetic engineering and tissue regeneration. The exploding field of stem cell research for reparative tissue and organ replacement demands an understanding of the morphogenetic mechanisms occurring during facial formation. The recipe for differentiation of stem cells in therapeutic cloning is similar to that of the pathways taken by the multilineage pluripotential cells of the early embryo. The same genes, growth factors, and signaling pathways that operate in the embryo are replicated in directed stem cell differentiation for therapeutic tissue replacement. Fundamental insights into pathophysiology, diseases, and dysmorphology are being revealed by molecular biology (Fig. 1.4).

The field of craniofacial embryology is currently undergoing a paradigmatic period of readjustment and discovery. The last decade has revealed a host of previously unknown factors in embryogenesis. During development, cells are monitored by genetically determined pathways and adjust their rates of accumulation, apoptosis, and hyperplasia to produce organs of predetermined size. The precise control of growth is of inestimable importance for, if each cell in our faces was to undergo just one more cell division, we would be horribly malformed.

The prior presence of the brain determines the subsequent development of the craniofacies. The rostral parts of the brain—the prosencephalon and mesencephalon—are specified by the orthodenticle homologues OTX1 and OTX2, while the HOX genes specify the rhombencephalon and establish spatial identity of prospective craniofacial compartments (Dixon et al. 2011; Larsen et al. 2010; Vieille-Grosjean et al. 1997). It is the brain underlying the future face that is a key component of cephalogenesis (Marcucio et al. 2011).