Small molecule inhibition of RAS/MAPK signaling ameliorates developmental pathologies of Kabuki Syndrome.

Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.

Retinoic Acid Maintains Function of Neural Crest-Derived Ocular and Craniofacial Structures in Adult Zebrafish.

Purpose: Retinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest-derived structures in adult zebrafish. Methods: Morphology and histology were analyzed by using live imaging, methylacrylate sections, and TUNEL assay. Functional analysis of vision and aqueous humor outflow were assayed with real-time imaging. Results: Both decreased and increased RA signaling altered craniofacial and ocular structures in adult zebrafish. Exogenous treatment with all-trans RA for 5 days resulted in a prognathic jaw, while inhibition of endogenous RA synthesis through treatment with 4-diethylaminobenzaldehyde (DEAB) decreased head height. In adult eyes, RA activity was localized to the retinal pigment epithelium, photoreceptors, outer plexiform layer, inner plexiform layer, iris stroma, and ventral canalicular network. Exogenous RA increased apoptosis in the iris stroma and canalicular network in the ventral iridocorneal angle, resulting in the loss of these structures and decreased aqueous outflow. DEAB, which decreased RA activity throughout the eye, induced widespread apoptosis, resulting in corneal edema, cataracts, retinal atrophy, and loss of iridocorneal angle structures. DEAB-treated fish were blind with no optokinetic response and no aqueous outflow from the anterior chamber. Conclusions: Tight control of RA levels is required for normal structure and function of the adult anterior segment. These studies demonstrated that RA plays an important role in maintaining ocular and craniofacial structures in adult zebrafish.

Pax3 and Pax7 play essential safeguard functions against environmental stress-induced birth defects.

Exposure to environmental teratogenic pollutant leads to severe birth defects. However, the biological events underlying these developmental abnormalities remain undefined. Here, we report a molecular link between an environmental stress response pathway and key developmental genes during craniofacial development. Strikingly, mutant mice with impaired Pax3/7 function display severe craniofacial defects. We show that these are associated with an upregulation of the signaling pathway mediated by the Aryl hydrocarbon receptor (AHR), the receptor to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), revealing a genetic interaction between Pax3 and AHR signaling. Activation of AHR signaling in Pax3-deficient embryos drives facial mesenchymal cells out of the cell cycle through the upregulation of p21 expression. Accordingly, inhibiting AHR activity rescues the cycling status of these cells and the facial closure of Pax3/7 mutants. Together, our findings demonstrate that the regulation of AHR signaling by Pax3/7 is required to protect against TCDD/AHR-mediated teratogenesis during craniofacial development.

Animal models for craniofacial reconstruction by stem/stromal cells.

The craniofacial region contains a variety of specified tissues, including bones, muscles, cartilages, teeth, blood vessels and nerves. Infections, traumas, genetic, anatomical, or congenital abnormalities could cause tissue defects in the region. Craniofacial tissue engineering and regeneration remain challenging problems for oral and maxillofacial surgeons and scientists. Stem cells isolated from the bone marrow, adipose tissue, dental pulp, the deciduous tooth, or the periodontium were proven to play an important role in tissue regeneration including craniofacial bone defect regeneration, facial nerve regeneration, TMJ (temporal-mandibular joint) condylar cartilage regeneration, TMJ disc regeneration and teeth regeneration in massive studies. In the review, the animal models for craniofacial engineering and regeneration are discussed. Specifically the modalities of establishing a defect model and treatment of the defect with various stem cells in combination with different cytokines and biomaterials are included. The review could be used to choose an appropriate experimental model for specific tissue defect, or to design innovative, reproducible, discriminative experimental models in the future.

Maternal diet supplementation with methyl donors and increased parity affect the incidence of craniofacial defects in the offspring of twisted gastrulation mutant mice.

Diets rich in methyl-donating compounds, including folate, can provide protection against neural tube defects, but their role in preventing craniofacial defects is less clear. Mice deficient in Twisted gastrulation (TWSG1), an extracellular modulator of bone morphogenetic protein signaling, manifest both midline facial defects and jaw defects, allowing study of the effects of methyl donors on various craniofacial defects in an experimentally tractable animal model. The goal of this study was to examine the effects of maternal dietary supplementation with methyl donors on the incidence and type of craniofacial defects among Twsg1(-/-) offspring. Nulliparous and primiparous female mice were fed an NIH31 standard diet (control) or a methyl donor supplemented (MDS) diet (folate, vitamin B-12, betaine, and choline). Observed defects in the pups were divided into those derived mostly from the first branchial arch (BA1) (micrognathia, agnathia, cleft palate) and midline facial defects in the holoprosencephaly spectrum (cyclopia, proboscis, and anterior truncation). In the first pregnancy, offspring of mice fed the MDS diet had lower incidence of BA1-derived defects (12.8% in MDS vs. 32.5% in control; P = 0.02) but similar incidence of midline facial defects (6.4% in MDS vs. 5.2% in control; P = 1.0). Increased maternal parity was independently associated with increased incidence of craniofacial defects after adjusting for diet (from 37.7 to 59.5% in control, P = 0.04 and from 19.1 to 45.3% in MDS, P = 0.045). In conclusion, methyl donor supplementation shows protective effects against jaw defects, but not midline facial defects, and increased parity can be a risk factor for some craniofacial defects.

Prevention of retinoic acid-induced early craniofacial abnormalities by vitamin B12 in mice.

OBJECTIVE: The purpose of the present study was to identify the potential effect of prenatal vitamin B12 administration on retinoic acid (RA)-induced early craniofacial abnormalities in mice and to investigate the possible mechanisms by which vitamin B12 reduces malformations. DESIGN: In our study, whole embryo culture was used to explore the effect of vitamin B12 on mouse embryos during the critical period of organogenesis. All embryos were exposed to 0.4 µM RA and different concentrations of vitamin B12 and scored for their growth in the branchial region at the end of a 48-hour culture period. The endothelin-1 (ET-1)/dHAND protein expression levels in the first branchial arch were investigated using an immunohistochemical method. RESULTS: In the whole embryo culture, 100 and 10 µM vitamin B12 dose-dependently prevented branchial region malformations and decreased craniofacial defects by 90.5% and 77.3%, respectively. ET-1 and dHAND protein levels were significantly increased in vitamin B12-supplemented embryos compared to the RA-exposed group in embryonic branchial region. CONCLUSIONS: These results suggest that vitamin B12 may prevent RA-induced craniofacial abnormalities via prevention of an RA-induced decrease of ET-1 and dHAND protein levels in the branchial region during the organogenic period. This study may shed new light on preventing craniofacial abnormalities.

Expansion and characterization of human embryonic stem cell-derived osteoblast-like cells.

Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies.

Introductory comments on special section-new developments in craniofacial biology: putting on a happy face.

Approximately three quarters of children with birth defects have anomalies that affect the craniofacial structures. Defects in this area of the body result in lifelong disability, major challenges to families and society and often a serious effect on life expectancy. Surgery has been the primary intervention for these disorders, with frequently less than optimal outcomes and risk for additional morbidity and mortality. The challenge for clinicians caring for these children is to develop new methods for the treatment and prevention of these disorders. An understanding of the evolution of the head and the finely tuned temporospatial signaling pathways involved is critical to understanding the origins of the vertebrates as well as of human craniofacial malformations. In the future, these new approaches will be based upon our enhanced understanding of the developmental tool kit fashioned by evolution and the application of this knowledge toward the development of new diagnostic, pharmacologic, and genetic interventions for these disorders.

[Advances in the study of the etiologic relationship between reduced folate carrier gene (RFC1) and neural tube and craniofacial defects].

Neural tube and craniofacial defects are common birth defects which are ascribed to the combination of genetic and environmental factors. The population epidemiological studies suggested that periconceptional use of multivitamins containing folic acid can reduce a woman's risk of having a child with neural tube and craniofacial defects. It's a major environmental factor that periconceptional women with deficiency of folic acid may increase their risk for delivering babies with neural tube and craniofacial defects, but the mechanism by which folic acid facilitates this risk reduction is unknown. This paper reviews folate transport carrier, Reduced Folate Carrier (RFC)'s characteristics in biological chemistry, physiological function, the folate transport mechanism, structure, function, regulation and expression of reduced folate carrier gene (RFC1), and the relationship between RFC1 with plasm or erythrocyte folate level and neural tube defects, et al. It is suggested a etiologic hypothesis in investigation of candidate gene encoding specific folate-related pathways of neural tube and craniofacial defects.

A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth.

The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation. PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors. To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene. The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth. The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors. However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences. Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis. In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mice.

New surgical technique for the correction of congenital muscular torticollis (wry neck).

Congenital muscular torticollis (wry neck) results from shortening of the sternocleidomastoid muscle and may lead to limitation of neck movement and craniofacial deformity. If conservative treatment is started early, with a regimen of passive stretching exercises and active strengthening of the contralateral muscle, about 95% of patients achieve an acceptable range of neck movement. The surgical management of patients who do not respond to physiotherapy remains controversial. Its aim is to provide a long-term, cosmetic restoration of neck mobility while minimizing the development of craniofacial deformity and upper cervical scoliosis; few previously advocated techniques achieve both these goals. We describe a technique that combines subperiosteal lengthening of the sternocleidomastoid muscle at its mastoid insertion, and division of lower fibrotic bands with minimal postoperative fibrosis. As the sternomastoid muscle is reattached lower down on the mastoid process, the lengthening of the muscle is stable, because the tendency to fibrosis and shortening is minimized. Comparison of the results with previous series shows that this technique provides immediate benefit and good long-term results.

Neural tube and craniofacial defects with special emphasis on folate pathway genes.

Neural tube and orofacial defects are common congenital malformations in humans. While etiologically heterogeneous, they are for the most part multifactorial in their pathogenesis, having both genetic and environmental components in their development. In recent years, there has been a great deal of epidemiologic evidence demonstrating that women who received multivitamins containing folic acid periconceptionally had significantly reduced occurrence and recurrence risks for producing infants with such malformations. This risk reduction is not observed in all populations, further suggestive of a genetic regulation of this phenomenon. Unfortunately, the mechanisms underlying the beneficial effects of folic acid are not well-understood. In this article, we review the relevant epidemiologic data on both neural tube defects and orofacial malformations, the fundamental embryological processes involved in closing the neural tube, and the development of the craniofacies, and propose a working hypothesis for susceptibility to these malformations. This hypothesis is based on the interworkings of cellular folate transport, focusing on the key elements involved in potocytosis. We propose that infants with mutations in the folate receptor alpha gene might be at increased risk for congenital anomalies due to a reduced binding affinity for 5-methyltetrahydrofolate, the physiologic form of folic acid. Various experimental approaches to test the working hypothesis are considered.