Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

3D atlas of the human fetal chondrocranium in the middle trimester.

The chondrocranium provides the key initial support for the fetal brain, jaws and cranial sensory organs in all vertebrates. The patterns of shaping and growth of the chondrocranium set up species-specific development of the entire craniofacial complex. The 3D development of chondrocranium have been studied primarily in animal model organisms, such as mice or zebrafish. In comparison, very little is known about the full 3D human chondrocranium, except from drawings made by anatomists many decades ago. The knowledge of human-specific aspects of chondrocranial development are essential for understanding congenital craniofacial defects and human evolution. Here advanced microCT scanning was used that includes contrast enhancement to generate the first 3D atlas of the human fetal chondrocranium during the middle trimester (13 to 19 weeks). In addition, since cartilage and bone are both visible with the techniques used, the  endochondral ossification of cranial base was mapped since this region is so critical for brain and jaw growth. The human 3D models are published as a scientific resource for human development.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral to calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. Time-lapse light-sheet imaging of mouse embryos revealed calvarial progenitors intercalate in 3D in the CM above the eye, and exhibit protrusive and crawling activity more apically. CM cells express non-canonical Wnt/planar cell polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand Wnt5a-/- mutants have less dynamic cell rearrangements and protrusive activity. Loss of CM-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of Osx+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

Perinatal imaging findings of a fetus with Pfeiffer syndrome and a heterozygous c.1019A>G, p.Tyr340Cys (Y340C) mutation in FGFR2 presenting a cloverleaf skull, craniosynostosis and short limbs on prenatal ultrasound mimicking thanatophoric dysplasia type II.

OBJECTIVE: We present perinatal imaging findings of a fetus with Pfeiffer syndrome and a heterozygous c.1019A>G, p.Tyr340Cys (Y340C) mutation in FGFR2 presenting a cloverleaf skull, craniosynostosis and short limbs on prenatal ultrasound mimicking thanatophoric dysplasia type II (TD2). CASE REPORT: A 37-year-old, gravida 2, para 1, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XY. However, craniofacial anomaly was found on prenatal ultrasound at 21 weeks of gestation, which showed a cloverleaf skull with severe craniosynostosis and relatively short straight long bones. Fetal magnetic resonance imaging (MRI) analysis at 22 weeks of gestation showed a cloverleaf skull, proptosis and relatively shallowing of the sylvian fissures. Prenatal ultrasound at 24 weeks of gestation showed a fetus with a cloverleaf skull with a biparietal diameter (BPD) of 6.16 cm (equivalent to 24 weeks), an abdominal circumference (AC) of 18.89 cm (equivalent to 24 weeks) and a femur length (FL) of 3.65 cm (equivalent to 21 weeks). A tentative diagnosis of TD2 was made. The pregnancy was subsequently terminated, and a 928-g malformed fetus was delivered with severe craniosynostosis, proptosis, midface retrusion, a cloverleaf skull, broad thumbs and broad big toes. The broad thumbs were medially deviated. Whole body X-ray showed a cloverleaf skull and straight long bones. However, molecular analysis of FGFR3 on the fetus revealed no mutation in the target regions. Subsequent whole exome sequencing (WES) on the DNA extracted from umbilical cord revealed a heterozygous c.1019A>G, p.Tyr340Cys (Y340C) mutation in the FGFR2 gene. CONCLUSION: Fetuses with a Y340C mutation in FGFR2 may present a cloverleaf skull on prenatal ultrasound, and WES is useful for a rapid differential diagnosis of Pfeiffer syndrome from TD2 under such a circumstance.

Key Roles of Gli1 and Ihh Signaling in Craniofacial Development.

The Hedgehog (Hh) signaling pathway orchestrates its influence through a dynamic interplay of Hh proteins, the cell surface receptor Ptch1, Smo, and Gli transcription factors, contributing to a myriad of developmental events. Indian Hedgehog (Ihh) and Gli zinc finger transcription factor 1 (Gli1) play crucial roles in developmental regulation within the Hh signaling pathway. Ihh regulates chondrocyte proliferation, differentiation, and bone formation, impacting the development of cranial bones, cartilage, and the temporomandibular joint (TMJ). Losing Ihh results in cranial bone malformation and decreased ossification and affects the formation of cranial base cartilage unions, TMJ condyles, and joint discs. Gli1 is predominantly expressed during early craniofacial development, and Gli1+ cells are identified as the primary mesenchymal stem cells (MSCs) for craniofacial bones, crucial for cell differentiation and morphogenesis. In addition, a complex mutual regulatory mechanism exists between Gli1 and Ihh, ensuring the normal function of the Hh signaling pathway by directly or indirectly regulating each other's expression levels. And the interaction between Ihh and Gli1 significantly impacts the normal development of craniofacial tissues. This review summarizes the pivotal roles of Gli1 and Ihh in the intricate landscape of mammalian craniofacial development and outlines the molecular regulatory mechanisms and intricate interactions governing the growth of bone and cartilage exhibited by Gli1 and Ihh, which provides new insights into potential therapeutic strategies for related diseases or researches of tissue regeneration.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Dynamic regulation and requirement for ribosomal RNA transcription during mammalian development.

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis-Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.

Ontogenetic variation in the skull of Stenopterygius quadriscissus with an emphasis on prenatal development.

The availability of a large sample size from a range of ontogenetic stages makes Stenopterygius quadriscissus a good model to study ontogenetic variation in a fossil sauropsid. We qualitatively examined pre- and postnatal ontogenetic changes in the cranium of S. quadriscissus. The prenatal ossification sequence is similar to other diapsids, exhibiting delayed chondrocranial ossification compared to the dermatocranium. In the dermatocranium, the circumorbital area is more ossified earlier in development relative to other elements, especially those of the skull roof where ossification is comparatively weaker across prenatal stages. Perinatally all cranial elements are ossified, and many scarf and step joints are already closed. We propose four prenatal and three postnatal stages in S. quadriscissus on the basis of relative ossification, size and qualitative cranial characters pertaining to the jugal, parietal, frontal, pterygoid and surangular. These will provide a basis for determining ontogenetic stages in other ichthyosaurs. Moreover, our postnatal observations aid in refining ontogenetic characters for phylogenetic studies. Lastly, we observed that the antimeric sutures of the midline of the skull roof are open perinatally and that fusion of the midline only appears in the adult stage. We hypothesize that the loose connection of the midline functions as a fontanelle, limiting potential damage during birth.

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.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

A comprehensive series of Irx cluster mutants reveals diverse roles in facial cartilage development.

Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.

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.

A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.

Essential function and targets of BMP signaling during midbrain neural crest delamination.

BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling.

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Constitutive expression of spliced XBP1 causes perinatal lethality in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor. XBP1S is a key mediator of the IRE1 branch that is essential for alleviating ER stress. We generated a novel mouse strain (referred to as "Xbp1CS/+ " mice) that constitutively expressed XBP1S after Cre recombinase-mediated recombination. Further breeding of these mice with Twist2 Cre recombinase (Twist2-Cre) knock-in mice generated Twist2-Cre;Xbp1CS/+ mice. Most Twist2-Cre;Xbp1CS/+ mice died shortly after birth. Reverse-transcription polymerase chain reaction (RT-PCR) showed that constitutive expression of XBP1S occurred in various mouse tissues examined, but not in the brain. Immunohistochemistry confirmed that although the immunostaining signals for total XBP1 (XBP1U and XBP1S) were found in the calvarial bones in both Twist2-Cre;Xbp1CS/+ and control mice, the signals for XBP1S were only detected in the Twist2-Cre;Xbp1CS/+ mice, but not in the control mice. These results suggest that a precise control of XBP1S production is essential for normal mouse development.

Craniofacial Development: Neural Crest in Molecular Embryology.

Craniofacial development, one of the most complex sequences of developmental events in embryology, features a uniquely transient, pluripotent stem cell-like population known as the neural crest (NC). Neural crest cells (NCCs) originate from the dorsal aspect of the neural tube and migrate along pre-determined routes into the developing branchial arches and frontonasal plate. The exceptional rates of proliferation and migration of NCCs enable their diverse contribution to a wide variety of craniofacial structures. Subsequent differentiation of these cells gives rise to cartilage, bones, and a number of mesenchymally-derived tissues. Deficiencies in any stage of differentiation can result in facial clefts and abnormalities associated with craniofacial syndromes. A small number of conserved signaling pathways are involved in controlling NC differentiation and craniofacial development. They are used in a reiterated fashion to help define precise temporospatial cell and tissue formation. Although many aspects of their cellular and molecular control have yet to be described, it is clear that together they form intricately integrated signaling networks required for spatial orientation and developmental stability and plasticity, which are hallmarks of craniofacial development. Mutations that affect the functions of these signaling pathways are often directly or indirectly identified in congenital syndromes. Clinical applications of NC-derived mesenchymal stem/progenitor cells, persistent into adulthood, hold great promise for tissue repair and regeneration. Realization of NCC potential for regenerative therapies motivates understanding of the intricacies of cell communication and differentiation that underlie the complexities of NC-derived tissues.

Fetal cranial growth trajectories are associated with growth and neurodevelopment at 2 years of age: INTERBIO-21st Fetal Study.

Many observational studies and some randomized trials demonstrate how fetal growth can be influenced by environmental insults (for example, maternal infections)1 and preventive interventions (for example, multiple-micronutrient supplementation)2 that can have a long-lasting effect on health, growth, neurodevelopment and even educational attainment and income in adulthood3. In a cohort of pregnant women (n = 3,598), followed-up between 2012 and 2019 at six sites worldwide4, we studied the associations between ultrasound-derived fetal cranial growth trajectories, measured longitudinally from <14 weeks' gestation, against international standards5,6, and growth and neurodevelopment up to 2 years of age7,8. We identified five trajectories associated with specific neurodevelopmental, behavioral, visual and growth outcomes, independent of fetal abdominal growth, postnatal morbidity and anthropometric measures at birth and age 2. The trajectories, which changed within a 20-25-week gestational age window, were associated with brain development at 2 years of age according to a mirror (positive/negative) pattern, mostly focused on maturation of cognitive, language and visual skills. Further research should explore the potential for preventive interventions in pregnancy to improve infant neurodevelopmental outcomes before the critical window of opportunity that precedes the divergence of growth at 20-25 weeks' gestation.

Delineating the roles of Grhl2 in craniofacial development through tissue-specific conditional deletion and epistasis approaches in mouse.

BACKGROUND: The highly conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. RESULTS: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/- ;Edn1+/- double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2aiCre ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. CONCLUSION: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non-cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung.

Heading for higher ground: Developmental origins and evolutionary diversification of the amniote face.

Amniotes, a clade of terrestrial vertebrates, which includes all of the descendants of the last common ancestor of the reptiles (including dinosaurs and birds) and mammals, is one of the most successful group of animals on our planet. In addition to having an egg equipped with an amnion, an adaptation to lay eggs on land, amniotes possess a number of other major morphological characteristics. Chief among them is the amniote skull, which can be classified into several major types distinguished by the presence and number of temporal fenestrae (windows) in the posterior part. Amniotes evolved from ancestors who possessed a skull composed of a complex mosaic of small bones separated by sutures. Changes in skull composition underlie much of the large-scale evolution of amniotes with many lineages showing a trend in reduction of cranial elements known as the "Williston's Law." The skull of amniotes is also arranged into a set of modules of closely co-evolving bones as revealed by modularity and integration tests. One of the most consistently recovered and at the same time most versatile modules is the "face," anatomically defined as the anterior portion of the head. The faces of amniotes display extraordinary amount of variation, with many adaptive radiations showing parallel tendencies in facial scaling, e.g., changes in length or width. This review explores the natural history of the amniote face and discusses how a better understanding of its anatomy and developmental biology helps to explain the outstanding scale of adaptive facial diversity. We propose a model for facial evolution in the amniotes, based on the differential rate of cranial neural crest cell proliferation and the timing of their skeletal differentiation.