What Do Animal Models Teach Us About Congenital Craniofacial Defects?

The formation of the head and face is a complex process which involves many different signaling cues regulating the migration, differentiation, and proliferation of the neural crest. This highly complex process is very error-prone, resulting in craniofacial defects in nearly 10,000 births in the United States annually. Due to the highly conserved mechanisms of craniofacial development, animal models are widely used to understand the pathogenesis of various human diseases and assist in the diagnosis and generation of preventative therapies and treatments. Here, we provide a brief background of craniofacial development and discuss several rare diseases affecting craniofacial bone development. We focus on rare congenital diseases of the cranial bone, facial jaw bones, and two classes of diseases, ciliopathies and RASopathies. Studying the animal models of these rare diseases sheds light not only on the etiology and pathology of each disease, but also provides meaningful insights towards the mechanisms which regulate normal development of the head and face.

Maternal choline supplementation mitigates alcohol-induced fetal cranio-facial abnormalities detected using an ultrasonographic examination in a sheep model.

Early detection of prenatal alcohol exposure is critical for designing and testing effectiveness of interventional therapeutics. Choline supplementation during and after prenatal alcohol exposure has shown promising benefits in improving outcomes in rodent models and clinical studies. A sheep model of first trimester-equivalent binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Pregnant sheep were randomly assigned to six groups: Saline + Placebo control, Saline + Choline, binge Alcohol + Placebo (light binging), binge Alcohol + Choline, Heavy binge Alcohol + Placebo (heavy binging), and Heavy binge Alcohol + Choline. Ewes received intravenous alcohol or saline on three consecutive days per week from gestation day (GD) 4-41 to mimic a first trimester-equivalent weekend binge-drinking paradigm. Choline (10 mg/kg in the daily food ration) was administered from GD 4 until term. On GD 76, 11 fetal ultrasonographic measurements were collected transabdominally. Heavy binge alcohol exposure reduced fetal Frontothalamic Distance (FTD), Mean Orbital Diameter (MOD), and Mean Lens Diameter (MLD), and increased Interorbital Distance (IOD) and Thalamic Width (TW). Maternal choline supplementation mitigated most of these alcohol-induced effects. Maternal choline supplementation also improved overall fetal femur and humerus bone lengths, compared to their respective placebo groups. Taken together, these results indicate a potential dose-dependent effect that could impact the sensitivity of these ultrasonographic measures in predicting prenatal alcohol exposure. This is the first study in the sheep model to identify biomarkers of prenatal alcohol exposure in utero with ultrasound and co-administration of maternal choline supplementation.

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.

Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD.

Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in ∼62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD.

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.

Pyridoxine might not have a preventive effect on the retinyl palmitate-induced viscerocranial anomalies.

Viscerocranial anomalies are induced in the presence of various teratogens. Vitamin A-induced cleft palate formation is one of the most frequently used experimental models in these studies. However, the underlying mechanisms are not yet fully understood. Several studies have shown that exogenous vitamin A disrupts the fusion of the palatal shelves by increasing the expression of epidermal growth factor receptor (EGFR). More recently, pyridoxine (vitamin B6) has been reported to have a potentially protective effect in regard to viscerocranial malformations. Therefore, in this study, we aimed to investigate whether pyridoxine has a preventive effect on retinyl palmitate-induced viscerocranial anomalies. The frequency of gross malformations induced by retinyl palmitate, the natural form of vitamin A, has been studied in a dose dependent manner. Low doses of retinyl palmitate (100 mg/kg) exposure on embryonic day (ED) 10 caused no gross anomalies in the rat fetuses. Teratogenic effects were observed only after exposure to higher dosages (1000 mg/kg) and primarily targeted the developing eyes and palates. On the other hand, co-administration of 10mg/kg pyridoxine, at ED 9 and 10, significantly increased the frequencies of anomalies, even in the moderate dosage (500 mg/kg) group. In all cleft palates, sustained expression of EGFR in the medial edge epithelium was detected by immunohistochemistry. These results show that co-administration of pyridoxine has an inductive rather than protective effect on the formation of viscerocranial malformations after exposure to hypervitaminosis-A.

Calcium folinate diminishes the teratogenic effects of all-trans retinoic acid in the developing craniofacial region and neural tube of the rat

This study tests the hypothesis that calcium folinate diminishes, or ameliorates, the teratogenic effects of all-trans retinoic acid during craniofacial and neural tube development. Four experimental groups were used; two were treated with three doses of 30mg/k.b.w all-trans retinoic acid (on gestational days 9, 10, and 11), with one of these groups having additional doses of calcium folinate (5mg/k.b.w.). The other two groups were given higher doses of all-trans retinoic acid (50mg/k.b.w), with again one of these groups having additional doses of calcium folinate (5mg/k.b.w.). Retinoic acid was administered by gastric intubation and calcium folinate by peritoneal injection. Two further control groups were used where the pregnant rats were exposed to neither all-trans retinoic acid nor calcium folinate. All rats were sacrificed on gestational day 18. Standard histological techniques were employed to assess the extent of abnormal development of the craniofacial region (including the palate) and the brain. The fetuses treated with all-trans retinoic acid alone showed varying degrees of neural tube defects (including excencephaly, myelomeningocele and spina bifida), eye malformations and clefting of the face and palate. However, fetuses treated with calcium folinate showed no neural tube and eye defects and only occasionally minor clefting in the presumptive hard palate. In addition, although many fetal absorptions and teratomas were seen within the litters of rats treated only with all-trans retinoic acid, there were no absorptions (and few teratomas) seen with calcium folinate supplements. The findings support our initial hypothesis concerning the beneficial effects of calcium folinate on craniofacial development (AU)

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Ethanol-mediated fetal dysmorphology and its relationship to the ontogeny of maternal liver metallothionein.

BACKGROUND: Fetal zinc (Zn) deficiency arising from ethanol-induction of the Zn-binding protein metallothionein (MT) in the mother's liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol-mediated fetal dysmorphology is also examined. METHODS: Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague-Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 microg Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. RESULTS: Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy-related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10-fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment. CONCLUSIONS: The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol-induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol-mediated teratogenicity.

Effect of different transfusion regimens on craniofacial appearance and dentition in severe thalassemic children.

Thalassemia is a group of inherited diseases with a defect in the synthesis of hemoglobin. Severe thalassemic subjects suffer from craniofacial deformities and malocclusion due to bone marrow hyperplasia compensating for ineffective erythropoiesis. Blood transfusions are used to maintain life and reduce complications. The transfusions may have benefits in reducing craniofacial and dentition abnormalities. However, appropriate therapy is still controversial. This study evaluated the effect of different transfusion regimens on craniofacial appearance and dentition. Ninety-two severe thalassemic patients, aged 6 -13 years, were divided into 3 groups according to the frequency of transfusion: 1) high transfusion: more than 12 times/year. 2) low transfusion: 6-12 times/year. 3) occasional transfusion: less than 5 times/year. The appearance and dentition were evaluated and compared among groups. Most subjects in the high transfusion group had a normal appearance and a class I molar and incisor relationship with normal overjet and overbite. More than half of subjects in the low and occasional groups showed craniofacial abnormalities and malocclusion, particular in the occasional group. Frequency of transfusion has an effect on craniofacial appearance and dental occlusion; only high frequent transfusions were effective in preventing craniofacial and dental defects.

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