IRF6 and SPRY4 Signaling Interact in Periderm Development.

Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the ß-catenin pathway.

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→ß-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects.

Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.

Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish.

Metallic nanoparticles such as nickel are used in catalytic sensing, and electronic applications, but health and environmental affects have not been fully investigated. While some metal nanoparticles result in toxicity, it is also important to determine whether nanoparticles of the same metal but of different size and shape changes toxicity. Three different size nickel nanoparticle (Ni NPs) of 30, 60, and 100 nm and larger particle clusters of aggregated 60 nm entities with a dendritic structure were synthesized and exposed to zebrafish embryos assessing mortality and developmental defects. Ni NPs exposure was compared to soluble nickel salts. All three 30, 60, and 100 nm Ni NPs are equal to or less toxic than soluble nickel while dendritic clusters were more toxic. With each Ni NP exposure, thinning of the intestinal epithelium first occurs around the LD10 continuing into the LD50. LD50 exposure also results in skeletal muscle fiber separation. Exposure to soluble nickel does not cause intestinal defects while skeletal muscle separation occurs at concentrations well over LD50. These results suggest that configuration of nanoparticles may affect toxicity more than size and defects from Ni NPs exposure occur by different biological mechanisms than soluble nickel.

Aryl hydrocarbon receptor-mediated down-regulation of sox9b causes jaw malformation in zebrafish embryos.

Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species, and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor, a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw after TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h after fertilization, and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4, and 12 h after exposure. Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting the formation of the embryonic jaw. Morpholino knockdown of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Loss of a single copy of the sox9b gene in sox9b(+/-) heterozygotes increased sensitivity to jaw malformation by TCDD. Finally, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation.

Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning.

We identified four insertional alleles of foxi one (foo), an embryonic lethal mutation in zebrafish that displays defects in both otic placode and the jaw. In foo/foo embryos the otic placode is split into two smaller placodes and mutant embryos show a dorsoventral (DV) cartilage defect manifested as a reduced hyomandibular and reduced third and fourth branchial arches. We identified foxi one (foo), the zebrafish ortholog of Foxi1 (FREAC6, FKHL10, HFH-3, Fkh10) and a member of the forkhead domain transcriptional regulator family, as the gene mutated in foo/foo embryos. foo is expressed in otic placode precursor cells, and foo/foo embryos lack placodal pax8 expression and have disorganized otic expression of pax2.1 and dlx3. Third stream neural crest cell migration, detected by dlx2 and krox20 expression, is aberrant in that it invades the otic placode territory. foo is expressed in pharyngeal pouch endoderm and is required for pouch expression of pax8 and proper patterning of other markers in the pouch such as nkx2.3. In foo/foo embryos, we observed a failure to maintain fgf3 expression in the pouches, followed by apoptosis of neural crest cells in adjacent arches. We conclude that foo expression is essential for pax8 expression probably downstream of Fgf signaling in a conserved pathway jointly required for integrity of patterning in the otic placode and pharyngeal pouches. We propose that correct placement of survival/proliferation cues is essential for shaping the pharyngeal cartilages and that evolutionary links between jaw and ear formation can be traced to Fgf-Foxi1-Pax8 pathways.

Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch.

In mammals, the first branchial arch (BA1) develops into a number of craniofacial skeletal elements including the jaws and teeth. Outgrowth and patterning of BA1 during early embryogenesis is thought to be controlled by signals from its covering ectoderm. Here we used Cre/loxP technology to inactivate the mouse Fgf8 gene in this ectoderm and have obtained genetic evidence that FGF8 has a dual function in BA1: it promotes mesenchymal cell survival and induces a developmental program required for BA1 morphogenesis. Newborn mutants lack most BA1-derived structures except those that develop from the distal-most region of BA1, including lower incisors. The data suggest that the BA1 primordium is specified into a large proximal region that is controlled by FGF8, and a small distal region that depends on other signaling molecules for its outgrowth and patterning. Because the mutant mice resemble humans with first arch syndromes that include agnathia, our results raise the possibility that some of these syndromes are caused by mutations that affect FGF8 signaling in BA1 ectoderm.

Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development.

The Msx1 homeobox gene is expressed at diverse sites of epithelial-mesenchymal interaction during vertebrate embryogenesis, and has been implicated in signalling processes between tissue layers. To determine the phenotypic consequences of its deficiency, we prepared mice lacking Msx1 function. All Msx1- homozygotes manifest a cleft secondary palate, a deficiency of alveolar mandible and maxilla and a failure of tooth development. These mice also exhibit abnormalities of the nasal, frontal and parietal bones, and of the malleus in the middle ear. Msx1 thus has a critical role in mediating epithelial-mesenchymal interactions during craniofacial bone and tooth development. The Msx1-/Msx1- phenotype is similar to human cleft palate, and provides a genetic model for cleft palate and oligodontia in which the defective gene is known.

Prenatal sonographic evidence supporting an in utero developmental etiology of Möbius sequence.

Möbius sequence features masklike facies with sixth and seventh nerve palsy and frequently micrognathia thought to result from a neuromuscular deficiency in early movement of the mandible. Failure to thrive in infancy is commonly due to feeding and aspiration difficulties. We present the first description of prenatal sonographic findings associated with this lesion, which also support an in utero developmental etiology of this rare condition.

Congenital oral synechiae.

Major congenital synechiae of the oral cavity constitute a clinically confusing spectrum of abnormalities. On the basis of clinical data, we propose two categories: 1) abnormalities secondary to persistence of the buccopharyngeal membrane and 2) abnormalities secondary to formation of ectopic membranes. An ectopic membrane results from abnormal fusion and can be subclassified as a subglossopalatal membrane, glossopalatal ankylosis, or syngnathia. This classification is supported by embryologic studies and is used to reclassify all cases reported since 1900. Distinct differences, such as the presence of associated limb anomalies, emerge; these are reviewed and add support to the proposed classification.

Cyclopia-otocephaly association: a new case of the most severe variant of agnathia-holoprosencephaly complex.

This report describes a new case of true cyclopia with otocephaly and additional brain malformations (alobar holoprosencephaly). This is a very rare occurrence involving lack of cleavage of the prosencephalon and disturbed development of the first branchial arch. An inductive defect of the prechordal mesoderm is considered as the cause for this malformation, which is a part of agnathia-holoprosencephaly complex.

Anophthalmos and first branchial arch defects.

The association of unilateral mandibulofacial dysostosis and anophthalmos at the same side is reported. The proband presented at the age of six months with: right anophthalmos and hypoplasia of the orbit at the same side, hypoplasia of the right mandible and maxilla, right external ear deformity, cyanotic heart disease, hemivertebrae, cataract and corneal opacity in the left eye. This case shows the maximal effect of the abnormal development of the first branchial arch (the mandible, maxilla, and ears) on the globe and the orbit. The other end of the spectrum in which the minimal effect on the globe was present (unilateral absence of choriocapillaris and retinal pigment epithelium inferiorly) was reported by Cotlier & Alghadyan in 1981. This supports the concept that abnormal development of the mandible may influence the development of the orbit and the globe. The extent of the influence of the abnormal developing first branchial arch on the eye is discussed.

[Embryoclinical correlation ofmaxillofacial abnormalities]. / Correlación embrioclínica de las malformaciones maxilofaciales.

Most publications on maxillofacial malformation are based on their topography and terminology. The list of authors is endless in most cases there are only minor variants. Thus, we are attempting to establish the embryoclinical correlation of maxillofacial malformations produced by changes in the structures of the cephalic pole of the embryo. For such systematization we have considered the following items: 1. Changes in the frontal structure. 2. Changes in upper maxillary structures. 3. Changes in the lower maxillary or mandibular structures. 4. Pathologic associations. In each of these buttons, the structures produced or correlated with them are subsequently analyzed, the same as basic changes and clinical syndromes produced by them and resulting in an embryoclinical systematization.

The adaptive mandible: a product of the relative osteo-neural growth.

The growth interrelation existing between the developing brain and its bony case (with the brain representing the morphogenetically determining structure to the shape and size of which the neurocranial bony envelope is adapted) is to be conceived just as a special case of a general phenomenon, the osteo-neural relation, which works throughout the entire vertebrate body. The morphological manifestations of this relation may be disclosed in the axial as well as in the appendicular skeleton. In the present communication the osteo-neural concept is applied to the morphogenesis of the facial skeleton with special reference to the mandible. The more or less elongated shape of the mandible in various species of vertebrates depends on the phylogenetically established higher or lesser degree of growth potentiality of the mandibular nerve. With the increasing size of the brain in the course of hominization the growth-in-length capability of the mandibular nerve is (compensatorily?) decreased with corresponding shortening of the mandible and with appearance of the chin. The proposed interpretation is supported by a model experiment. The highly characteristic shortening and angulation of the lower beak which may be produced experimentally in the chick embryo by a great variety of teratogens, is related most probably to the same underlying mechanism, viz., to the primary inhibition of the highly susceptible neural growth with the secondary adaptive deformity of the beak. Pierre-Robin-Syndrome (cleft palate and micrognathia) may be readily explained by a growth insufficiency of the palatine and mandibular nerves.

[Branchiogenic preauricular fistulae]. / Uber branchiogene präaurikuläre Fisteln.

Seventeen patients with dimples, fistulae and cysts in the region of the cheek and the preauricular field are reported. All are located in the neighborhood of the dorsal end of the first visceral arch. Both the fistulae of the ascending helix and the upper preauricular fistulae must be regarded as relics of the dorsal end of the first branchial cleft. This interpretation supports the theory of Wood-Jones and I-Chuan according to which the tragus is formed from the material of the mandibular arch, and the remaining part of the auricle from the mesoderm of the second visceral arch. Similar anomalies in the neighborhood of the angle of the mouth, the extraoral opening of an accessory duct of Stensen and the inferior preauricular fistula are sufficiently explained by the incomplete closure or tearing of the embryonal oral aperture.