Augmentation of BMP Signaling in Cranial Neural Crest Cells Leads to Premature Cranial Sutures Fusion through Endochondral Ossification in Mice.

Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Embryonic requirements for Tcf12 in the development of the mouse coronal suture.

A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in either of the bHLH transcription factors TWIST1 and TCF12. Although compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 had remained unexplored. Here, we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in embryonic neural crest and mesoderm, but not in postnatal suture mesenchyme, disrupts the coronal suture. We also detected asymmetric distribution of mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry is associated with bones meeting end-on-end, possibly contributing to synostosis. Our results support embryonic requirements of Tcf12 in proper formation of the overlapping coronal suture.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Resolving homology in the face of shifting germ layer origins: Lessons from a major skull vault boundary.

The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent 'coronal' suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.

Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome.

Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.

Culturing and Manipulation of O9-1 Neural Crest Cells.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear.

Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.

Yap and Taz play a crucial role in neural crest-derived craniofacial development.

The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1(Cre) and Wnt1(Cre2SOR) drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1(Cre2SOR) mutants had an open cranial neural tube phenotype that was not evident in Wnt1(Cre) mutants. In O9-1 CNC cells, the loss of Yap impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy-Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-PCR experiments supported the conclusion that Foxc1 is directly regulated by the Yap-Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.

The Development of the Calvarial Bones and Sutures and the Pathophysiology of Craniosynostosis.

The skull vault is a complex, exquisitely patterned structure that plays a variety of key roles in vertebrate life, ranging from the acquisition of food to the support of the sense organs for hearing, smell, sight, and taste. During its development, it must meet the dual challenges of protecting the brain and accommodating its growth. The bones and sutures of the skull vault are derived from cranial neural crest and head mesoderm. The frontal and parietal bones develop from osteogenic rudiments in the supraorbital ridge. The coronal suture develops from a group of Shh-responsive cells in the head mesoderm that are collocated, with the osteogenic precursors, in the supraorbital ridge. The osteogenic rudiments and the prospective coronal suture expand apically by cell migration. A number of congenital disorders affect the skull vault. Prominent among these is craniosynostosis, the fusion of the bones at the sutures. Analysis of the pathophysiology underling craniosynostosis has identified a variety of cellular mechanisms, mediated by a range of signaling pathways and effector transcription factors. These cellular mechanisms include loss of boundary integrity, altered sutural cell specification in embryos, and loss of a suture stem cell population in adults. Future work making use of genome-wide transcriptomic approaches will address the deep structure of regulatory interactions and cellular processes that unify these seemingly diverse mechanisms.

Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis.

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

A stable cranial neural crest cell line from mouse.

Cranial neural crest cells give rise to ectomesenchymal derivatives such as cranial bones, cartilage, smooth muscle, dentin, as well as melanocytes, corneal endothelial cells, and neurons and glial cells of the peripheral nervous system. Previous studies have suggested that although multipotent stem-like cells may exist during the course of cranial neural crest development, they are transient, undergoing lineage restriction early in embryonic development. We have developed culture conditions that allow cranial neural crest cells to be grown as multipotent stem-like cells. With these methods, we obtained 2 independent cell lines, O9-1 and i10-1, which were derived from mass cultures of Wnt1-Cre; R26R-GFP-expressing cells. These cell lines can be propagated and passaged indefinitely, and can differentiate into osteoblasts, chondrocytes, smooth muscle cells, and glial cells. Whole-genome expression profiling of O9-1 cells revealed that this line stably expresses stem cell markers (CD44, Sca-1, and Bmi1) and neural crest markers (AP-2α, Twist1, Sox9, Myc, Ets1, Dlx1, Dlx2, Crabp1, Epha2, and Itgb1). O9-1 cells are capable of contributing to cranial mesenchymal (osteoblast and smooth muscle) neural crest fates when injected into E13.5 mouse cranial tissue explants and chicken embryos. These results suggest that O9-1 cells represent multipotent mesenchymal cranial neural crest cells. The O9-1 cell line should serve as a useful tool for investigating the molecular properties of differentiating cranial neural crest cells.

Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells.

The Notch pathway is crucial for a wide variety of developmental processes including the formation of tissue boundaries. That it may function in calvarial suture development and figure in the pathophysiology of craniosynostosis was suggested by the demonstration that heterozygous loss of function of JAGGED1 in humans can cause Alagille syndrome, which has craniosynostosis as a feature. We used conditional gene targeting to examine the role of Jagged1 in the development of the skull vault. We demonstrate that Jagged1 is expressed in a layer of mesoderm-derived sutural cells that lie along the osteogenic-non-osteogenic boundary. We show that inactivation of Jagged1 in the mesodermal compartment of the coronal suture, but not in the neural crest compartment, results in craniosynostosis. Mesodermal inactivation of Jagged1 also results in changes in the identity of sutural cells prior to overt osteogenic differentiation, as well as defects in the boundary between osteogenic and non-osteogenic compartments at the coronal suture. These changes, surprisingly, are associated with increased expression of Notch2 and the Notch effector, Hes1, in the sutural mesenchyme. They are also associated with an increase in nuclear ß-catenin. In Twist1 mutants, Jagged1 expression in the suture is reduced substantially, suggesting an epistatic relationship between Twist1 and Jagged1. Consistent with such a relationship, Twist1-Jagged1 double heterozygotes exhibit a substantial increase in the severity of craniosynostosis over individual heterozygotes. Our results thus suggest that Jagged1 is an effector of Twist1 in coronal suture development.

Inactivation of Msx1 and Msx2 in neural crest reveals an unexpected role in suppressing heterotopic bone formation in the head.

In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.

EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis.

Heterozygous loss of Twist1 function causes coronal synostosis in both mice and humans. We showed previously that in mice this phenotype is associated with a defect in the neural crest-mesoderm boundary within the coronal suture, as well as with a reduction in the expression of ephrin A2 (Efna2), ephrin A4 (Efna4) and EphA4 in the coronal suture. We also demonstrated that mutations in human EFNA4 are a cause of non-syndromic coronal synostosis. Here we investigate the cellular mechanisms by which Twist1, acting through Eph-ephrin signaling, regulates coronal suture development. We show that EphA4 mutant mice exhibit defects in the coronal suture and neural crest-mesoderm boundary that phenocopy those of Twist1(+/-) mice. Further, we demonstrate that Twist1 and EphA4 interact genetically: EphA4 expression in the coronal suture is reduced in Twist1 mutants, and compound Twist1-EphA4 heterozygotes have suture defects of greater severity than those of individual heterozygotes. Thus, EphA4 is a Twist1 effector in coronal suture development. Finally, by DiI labeling of migratory osteogenic precursor cells that contribute to the frontal and parietal bones, we show that Twist1 and EphA4 are required for the exclusion of such cells from the coronal suture. We suggest that the failure of this process in Twist1 and EphA4 mutants is the cause of craniosynostosis.

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development.

UNLABELLED: The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing beta-galactosidase under the control of Msx2 promoter by fluorescence-activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing alpha-smooth muscle actin (alpha-SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet-derived growth factor receptor alpha. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki-67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti-ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM(+) cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM(+) cells rapidly acquired myofibroblastic morphology and alpha-SMA expression. The ALCAM(+) cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM(+) cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1-Cre and ROSA26 reporter mice. CONCLUSION: Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM(+) submesothelial cells may be a precursor for HSCs in developing liver.

Germline competent embryonic stem cells derived from rat blastocysts.

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

Msx1 and Msx2 are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium.

BACKGROUND: Msx1 and Msx2, which belong to the highly conserved Nk family of homeobox genes, display overlapping expression patterns and redundant functions in multiple tissues and organs during vertebrate development. Msx1 and Msx2 have well-documented roles in mediating epithelial-mesenchymal interactions during organogenesis. Given that both Msx1 and Msx2 are crucial downstream effectors of Bmp signaling, we investigated whether Msx1 and Msx2 are required for the Bmp-induced endothelial-mesenchymal transformation (EMT) during atrioventricular (AV) valve formation. RESULTS: While both Msx1-/- and Msx2-/- single homozygous mutant mice exhibited normal valve formation, we observed hypoplastic AV cushions and malformed AV valves in Msx1-/-; Msx2-/- mutants, indicating redundant functions of Msx1 and Msx2 during AV valve morphogenesis. In Msx1/2 null mutant AV cushions, we found decreased Bmp2/4 and Notch1 signaling as well as reduced expression of Has2, NFATc1 and Notch1, demonstrating impaired endocardial activation and EMT. Moreover, perturbed expression of chamber-specific genes Anf, Tbx2, Hand1 and Hand2 reveals mispatterning of the Msx1/2 double mutant myocardium and suggests functions of Msx1 and Msx2 in regulating myocardial signals required for remodelling AV valves and maintaining an undifferentiated state of the AV myocardium. CONCLUSION: Our findings demonstrate redundant roles of Msx1 and Msx2 in regulating signals required for development of the AV myocardium and formation of the AV valves.

Concerted action of Msx1 and Msx2 in regulating cranial neural crest cell differentiation during frontal bone development.

The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue-tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, we generated Msx1(-/-);Msx2(-/-) mice. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, our data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway.

Msx1 and Msx2 regulate survival of secondary heart field precursors and post-migratory proliferation of cardiac neural crest in the outflow tract.

Msx1 and Msx2 are highly conserved, Nk-related homeodomain transcription factors that are essential for a variety of tissue-tissue interactions during vertebrate organogenesis. Here we show that combined deficiencies of Msx1 and Msx2 cause conotruncal anomalies associated with malalignment of the cardiac outflow tract (OFT). Msx1 and Msx2 play dual roles in outflow tract morphogenesis by both protecting secondary heart field (SHF) precursors against apoptosis and inhibiting excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the conotruncal cushions. During incorporation of SHF precursors into the OFT myocardium, ectopic apoptosis in the Msx1-/-; Msx2-/- mutant SHF is associated with reduced expression of Hand1 and Hand2, which from work on Hand1 and Hand2 mutants may be functionally important in the inhibition of apoptosis in Msx1/2 mutants. Later during aorticopulmonary septation, excessive proliferation in the OFT cushion mesenchyme and myocardium of Msx1-/-; Msx2-/- mutants is associated with premature down-regulation of p27(KIP1), an inhibitor of cyclin-dependent kinases. Diminished accretion of SHF precursors to the elongating OFT myocardium and excessive accumulation of mesenchymal cells in the conotruncal cushions may work together to perturb the rotation of the truncus arteriosus, leading to OFT malalignment defects including double-outlet right ventricle, overriding aorta and pulmonary stenosis.

Recent advances in craniofacial morphogenesis.

Craniofacial malformations are involved in three fourths of all congenital birth defects in humans, affecting the development of head, face, or neck. Tremendous progress in the study of craniofacial development has been made that places this field at the forefront of biomedical research. A concerted effort among evolutionary and developmental biologists, human geneticists, and tissue engineers has revealed important information on the molecular mechanisms that are crucial for the patterning and formation of craniofacial structures. Here, we highlight recent advances in our understanding of evo-devo as it relates to craniofacial morphogenesis, fate determination of cranial neural crest cells, and specific signaling pathways in regulating tissue-tissue interactions during patterning of craniofacial apparatus and the morphogenesis of tooth, mandible, and palate. Together, these findings will be beneficial for the understanding, treatment, and prevention of human congenital malformations and establish the foundation for craniofacial tissue regeneration.