Medial Process Research Articles

Cilia-dependent GLI processing in neural crest cells is required for tongue development

Ciliopathies are a class of diseases caused by the loss of a ubiquitous, microtubule-based organelle called a primary cilium. Ciliopathies commonly result in defective development of the craniofacial complex, causing midfacial defects, craniosynostosis, micrognathia and aglossia. Herein, we explored how the conditional loss of primary cilia on neural crest cells (Kif3af/f;Wnt1-Cre) generated aglossia. On a cellular level, our data revealed that aglossia in Kif3af/f;Wnt1-Cre embryos was due to a loss of mesoderm-derived muscle precursors migrating into and surviving in the tongue anlage. To determine the molecular basis for this phenotype, we performed RNA-seq, in situ hybridization, qPCR and Western blot analyses. We found that transduction of the Sonic hedgehog (Shh) pathway, rather than other pathways previously implicated in tongue development, was aberrant in Kif3af/f;Wnt1-Cre embryos. Despite increased production of full-length GLI2 and GLI3 isoforms, previously identified GLI targets important for mandibular and glossal development (Foxf1, Foxf2, Foxd1 and Foxd2) were transcriptionally downregulated in Kif3af/f;Wnt1-Cre embryos. Genetic removal of GLI activator (GLIA) isoforms in neural crest cells recapitulated the aglossia phenotype and downregulated Fox gene expression. Genetic addition of GLIA isoforms in neural crest cells partially rescued the aglossia phenotype and Fox gene expression in Kif3af/f;Wnt1-Cre embryos. Together, our data suggested that glossal development requires primary cilia-dependent GLIA activity in neural crest cells. Furthermore, these data, in conjunction with our previous work, suggested prominence specific roles for GLI isoforms; with development of the frontonasal prominence relying heavily on the repressor isoform and the development of the mandibular prominence/tongue relying heavily on the activator isoform.

Columellar Reconstruction: A New Technique for Median Cleft Lip Repair.

Median cleft lip (MCL), failure of the fusion of the medial nasal prominences in the midline resulting in vertical cleft through the center of the upper lip, can be repaired by various columellar reconstruction approaches depending on the nature of patient. The main drawback of these surgical techniques is the use of healthy neighboring structures that may compromise the process. In this report, the authors present a new approach for repairing MCL which involves simultaneous columellar reconstruction without damaging any nasal units. Briefly, columella was reconstructed with 2 lateral triangular flaps harvested from both cleft sides. They were sutured together and attached to the ventral surface of the nasal dome at the midline. The procedure was resulted in satisfactory clinical and esthetic outcome suggesting this approach may present a better alternative for this condition.

Full moon shaped unusual appearance of a globulomaxillary cyst: A case report

Globulomaxillary cyst is a rare, spurious cyst. It was considered to be an inclusion or developmental cyst that arises from entrapped nonodontogenic epithelium in the globulomaxillary suture. Later, it was included as a fissural cyst secondary to proliferation of entrapped epithelium between the globular portion of the medial nasal and maxillary processes. Because of lack of evidence to support the theory of embryogenic epithelial entrapment in the site, most authors dispute the presence of the lesion. Herein, we are presenting a case of globulomaxillary cyst in a 20 year old female patient with an unusual radiographic appearance as a full moon.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development.

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

A Congenital Alar Defect and Its Reconstruction

The development of the face occurs mainly between the fourth and eighth weeks. By the end of the fourth week, bilateral oval thickenings of the surface ectoderm, called nasal placodes, develop on each side of the inferior part of the frontonasal prominence. The frontonasal prominence forms the forehead, dorsum of the nose, and apex of the nose. The alae of the nose are derived from the lateral nasal prominences (1).

Globulomaxillary Cyst in a 16 year old male patient-A case report with review

Globulomaxillary cyst is an uncommon, specious cyst. It was traditionally described as a fissural cyst between the maxillary lateral incisor and canine teeth, secondary to proliferation of entrapped epithelium between the globular portion of the medial nasal and maxillary processes. They are painless, submucosal, non-odontogenic jaw cysts presenting as soft tissue swellings in the maxillary anterior mucolabial fold lateral to midline. Present case documents 16 year old male patient with diffuse extra-oral swelling present involving the philtrum and extending upto the nasolabial fold was observed on the left side of the face. Intraorally well-defined soft and fluctuant swelling was present with pus discharge. Radiographically, well defined heart shaped radiolucency was observed in the left maxillary periapical region. The cyst was enucleated using intra-oral approach. Histopathological findings revealed pseudostratified ciliated epithelium with overlying inflammatory cells and goblet cells. Diagnosis of globulomaxillary cyst was confirmed.

Tessier No. 3 incomplete cleft: Nasal reconstruction using turbinate mucosal flap

Facial clefts are extremely rare congenital deformities, especially Type 3. In 1976, Tessier classified the clefts between 0 and 14 based on the central facial landmarks. The Tessier type 3 facial cleft, also called a nasoocular or a nasomaxillary cleft, results from the disruption of the lateral nasal and maxillary processes. It is characterized by inferior displacement of the medial canthus, superior displacement of the alar base, cleft lip and palate, coloboma of lower eyelids, nasolacrimal abnormalities, cleft of the inferomedial orbital wall, and teleorbitism. Although the literature is replete with classification and morbid anatomy of such cases, surgical management is still a challenge. In this article, we report a Tessier No. 3 incomplete cleft, wherein the turbinate mucosal flap was used for the nasal lining in reconstruction of the nose. Probably, this is the first time that the turbinate mucosal flap is used for reconstruction in Tessier No. 3 cleft.

Molecular mechanisms of midfacial developmental defects.

The morphogenesis of midfacial processes requires the coordination of a variety of cellular functions of both mesenchymal and epithelial cells to develop complex structures. Any failure or delay in midfacial development as well as any abnormal fusion of the medial and lateral nasal and maxillary prominences will result in developmental defects in the midface with a varying degree of severity, including cleft, hypoplasia, and midline expansion. Despite the advances in human genome sequencing technology, the causes of nearly 70% of all birth defects, which include midfacial development defects, remain unknown. Recent studies in animal models have highlighted the importance of specific signaling cascades and genetic-environmental interactions in the development of the midfacial region. This review will summarize the current understanding of the morphogenetic processes and molecular mechanisms underlying midfacial birth defects based on mouse models with midfacial developmental abnormalities.

Dual embryonic origin of maxillary lateral incisors: clinical implications in patients with cleft lip and palate.

Introduction: Cleft lip and palate are craniofacial anomalies highly prevalent in the overall population. In oral clefts involving the alveolar ridge, variations of number, shape, size and position are observed in maxillary lateral incisors. The objective of this manuscript is to elucidate the embryonic origin of maxillary lateral incisors in order to understand the etiology of these variations. Contextualization: The hypothesis that orofacial clefts would split maxillary lateral incisor buds has been previously reported. However, recent studies showed that maxillary lateral incisors have dual embryonic origin, being partially formed by both the medial nasal process and the maxillary process. In other words, the mesial half of the lateral incisor seems to come from the medial nasal process while the distal half of the lateral incisor originates from the maxillary process. In cleft patients, these processes do not fuse, which results in different numerical and positional patterns for lateral incisors relating to the alveolar cleft. In addition to these considerations, this study proposes a nomenclature for maxillary lateral incisors in patients with cleft lip and palate, based on embryology and lateral incisors position in relation to the alveolar cleft. Conclusion: Embryological knowledge on the dual origin of maxillary lateral incisors and the use of a proper nomenclature for their numerical and positional variations renders appropriate communication among professionals and treatment planning easier, in addition to standardizing research analysis.

Surgical repair of the isolated incomplete median cleft lip

Median cleft lip refers to a vertical cleft on the midline of the upper lip. It is a rare congenital craniofacial anomaly brought about by a fusion failure in the medial nasal prominence. A novel surgical approach to median cleft lips and their repair is reported herein, with reference to a clinical case. The patient had a cleft in the lower half of the upper lip. There were no other craniofacial anomalies in this patient other than the cleft. Within the framework of the patient's surgical treatment, a functional and cosmetically satisfactory result was achieved by performing a V–Y advancement flap on the columella base, Z-plasty in the vermillion zone, and Z pattern muscular tissue repair, without having to resort to any tissue excisions.

Recent insights into the morphological diversity in the amniote primary and secondary palates.

The assembly of the upper jaw is a pivotal moment in the embryonic development of amniotes. The upper jaw forms from the fusion of the maxillary, medial nasal, and lateral nasal prominences, resulting in an intact upper lip/beak and nasal cavities; together called the primary palate. This process of fusion requires a balance of proper facial prominence shape and positioning to avoid craniofacial clefting, whilst still accommodating the vast phenotypic diversity of adult amniotes. As such, variation in craniofacial ontogeny is not tolerated beyond certain bounds. For clarity, we discuss primary palatogenesis of amniotes into in two categories, according to whether the nasal and oral cavities remain connected throughout ontogeny or not. The transient separation of these cavities occurs in mammals and crocodilians, while remaining connected in birds, turtles and squamates. In the latter group, the craniofacial prominences fuse around a persistent choanal groove that connects the nasal and oral cavities. Subsequently, all lineages except for turtles, develop a secondary palate that ultimately completely or partially separates oral and nasal cavities. Here, we review the shared, early developmental events and highlight the points at which development diverges in both primary and secondary palate formation.

Sox10ER(T2) CreER(T2) mice enable tracing of distinct neural crest cell populations.

Neural crest cells play an important role in craniofacial morphogenesis and many other developmental processes. The formation of neural crest cells (NCCs) in vivo is a highly dynamic process and remains to be fully understood. To investigate the spatiotemporal patterning of NCCs in vivo, we have generated Sox10ER(T2) CreER(T2) (SECE) mice, a transgenic line driving inducible Cre expression in NCCs. Inducing Cre activity at different stages triggered reporter expression in distinct NCC populations in SECE; R26R mice. By optimizing the timing and dosage of tamoxifen administration, we controlled Cre expression specifically in cranial NCCs. Using this approach, we demonstrate an important role for PDGFRα in cranial NCCs mitosis within the mandibular processes. Further reducing Cre activity within the cranial NCCs of SECE; R26R embryos revealed that SECE labels preferentially progenitors of medial nasal process (MNP) rather than the lateral nasal process (LNP), before their formation from the frontonasal prominence (FNP). Our results indicate that NCCs are formed sequentially from rostral to caudal regions along the neural tube. These findings also suggest that NCCs within the FNP become specified regionally and genetically before they divide into MNP and LNP.

Isolated Congenital Nasal Bifid Septum Separated by a Wide Layer of Soft Tissue.

Isolated Congenital Nasal Bifid Septum Separated by a Wide Layer of Soft Tissue.

Divergence of craniofacial developmental trajectories among avian embryos.

Morphological divergence among related species involves changes to developmental processes. When such variation arises in development has garnered considerable theoretical interest relating to the broader issue of how development may constrain evolutionary change. The hourglass model holds that while early developmental events may be highly evolvable, there is a phylotypic stage when key developmental events are conserved. Thus, evolutionary divergence among related species should tend to arise after such a stage of reduced evolvability and, consequently, reduced variation among species. We test this prediction by comparing developmental trajectories among three avian species of varying relatedness (chick, quail, and duck) to locate their putative point of divergence. Three-dimensional geometric morphometrics and trajectory analyses were used to measure the significance of the facial shape variation observed among these species. Duck embryos, being more distantly related, differed from the more closely-related chick and quail embryos in the enlargement of their frontonasal prominences. Phenotypic trajectory analyses demonstrated divergence of the three species, most notably, duck. The results demonstrate that the two more closely related species share similar facial morphologies for a longer time during development, while ducks diverge. This suggests a surprising lability of craniofacial development during early face formation. Developmental Dynamics 244:1158-1167, 2015. © 2015 Wiley Periodicals, Inc.

Diversity in primary palate ontogeny of amniotes revealed with 3D imaging

The amniote primary palate encompasses the upper lip and the nasal cavities. During embryonic development, the primary palate forms from the fusion of the maxillary, medial nasal and lateral nasal prominences. In mammals, as the primary palate fuses, the nasal and oral cavities become completely separated. Subsequently, the tissue demarcating the future internal nares (choanae) thins and becomes the bucconasal membrane, which eventually ruptures and allows for the essential connection of the oral and nasal cavities to form. In reptiles (including birds), the other major amniote group, primary palate ontogeny is poorly studied with respect to prominence fusion, especially the formation of a bucconasal membrane. Using 3D optical projection tomography, we found that the prominences that initiate primary palate formation are similar between mammals and crocodilians but distinct from turtles and lizards, which are in turn similar to each other. Chickens are distinct from all non-avian lineages and instead resemble human embryos in this aspect. The majority of reptiles maintain a communication between the oral and nasal cavities via the choanae during primary palate formation. However, crocodiles appear to have a transient separation between the oral and nasal cavities. Furthermore, the three lizard species examined here, exhibit temporary closure of their external nares via fusion of the lateral nasal prominences with the frontonasal mass, subsequently reopening them just before hatching. The mechanism of the persistent choanal opening was examined in chicken embryos. The mesenchyme posterior/dorsal to the choana had a significant decline in proliferation index, whereas the mesenchyme of the facial processes remained high. This differential proliferation allows the choana to form a channel between the oral and nasal cavities as the facial prominences grow and fuse around it. Our data show that primary palate ontogeny has been modified extensively to support the array of morphological diversity that has evolved among amniotes.

Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate.

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15th to 24th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.

Management of Midline Facial Clefts.

Median or midline facial clefts are rare anomalies of developmental origin, etiology of whose occurrence is still unknown precisely. The most basic presentation of midline facial clefts is in the form of a Median cleft lip which is defined as any congenital vertical cleft through the centre of the upper lip. First described by Bechard in 1823, it is the most common amongst all atypical clefts reported. The incidence is about 1:10,00,000 births. This may occur as a sporadic event or as a part of an inherited sequence of anomalies. It arises embryologically from incomplete fusion of the medial nasal prominences. The authors present a series of eight cases with varying degrees of midline facial clefts. This review article aims to give a broad idea on the various classifications used for further understanding of midline facial clefts and a brief idea about the various surgical management techniques used in the repair of these facial clefts.

Patterning of mammalian heterodont dentition within the upper and lower jaws.

Mammalian heterodont dentition is differentiated into incisors, canines, premolars, and molars in the mesial-distal direction, in both the upper and lower jaws. Although all the lower teeth are rooted in the mandible, the upper incisors are rooted in the premaxilla and the upper canine and the teeth behind it are in the maxilla. The present study uncovers ontogenetic backgrounds to these shared and differing mesiodistal patterns of the upper and lower dentition. To this end, we examined the dentition development of the house shrew, Suncus murinus, instead of the rodent model animals because the dentition of this primitive eutherian species includes all the tooth classes, and no toothless diastema region. In the shrew, the upper incisor-forming region extended over the medial nasal prominence and the mesial part of the maxillary prominence. Consequently, the maxillary and mandibular prominences were in a mirror-image relationship in terms of the mesiodistally differentiated tooth-forming regions and of the complementary gene expression pattern, with Bmp4 in the mesial and Fgf8 in the distal regions. This suggests shared molecular mechanisms regulating tooth class differentiation between the upper and lower jaws. However, the premaxillary bone appeared within the mesenchyme of the medial nasal prominence, but grew distally beyond the former epithelial boundary with the maxillary prominence to form, finally, the incisive (premaxillary-maxillary) suture just mesial to the canine. Therefore, the developmental locations of the upper incisors are not inconsistent with the classical osteological criterion of the upper canine by comparative odontologists.

A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face

The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes.