The Effect of Liposomal Curcumin as an Anti-Inflammatory Strategy on Lipopolysaccharide e from Porphyromonas gingivalis Treated Endothelial Committed Neural Crest Derived Stem Cells: Morphological and Molecular Mechanisms.

Curcumin, a yellow polyphenol extracted from the turmeric root is used as a diet supplement. It exhibits anti-inflammatory, antioxidant, and antitumor properties by modulating different intracellular mechanisms. Due to their low solubility in water, the curcumin molecules must be encapsulated into liposomes to improve the bioavailability and biomedical potential. For the periodontal tissue and systemic health, it is essential to regulate the local inflammatory response. In this study, the possible beneficial effect of liposomes loaded with curcumin (CurLIP) in neural crest-derived human periodontal ligament stem cells (hPDLSCs) and in endothelial-differentiated hPDLSCs (e-hPDLSCs) induced with an inflammatory stimulus (lipopolysaccharide obtained from Porphyromonas gingivalis, LPS-G) was evaluated. The CurLIP formulation exhibited a significant anti-inflammatory effect by the downregulation of Toll-like receptor-4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa light chain enhancer of activated B cells (NFkB)/NLR Family Pyrin Domain Containing 3 (NLRP3)/Caspase-1/Interleukin (IL)-1ß inflammation cascade and reactive oxygen species (ROS) formation. Moreover, the exposure to LPS-G caused significant alterations in the expression of epigenetic modifiers, such as DNA Methyltransferase 1 (DNMT1) and P300, while the CurLIP treatment showed physiological expression. Overall, our in vitro study provides novel mechanistic insights into the intracellular pathway exert by CurLIP in the regulation of inflammation and epigenetic modifications.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis.

The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development.

Roles for FGF in lamprey pharyngeal pouch formation and skeletogenesis highlight ancestral functions in the vertebrate head.

A defining feature of vertebrates (craniates) is a pronounced head supported and protected by a cellularized endoskeleton. In jawed vertebrates (gnathostomes), the head skeleton is made of rigid three-dimensional elements connected by joints. By contrast, the head skeleton of modern jawless vertebrates (agnathans) consists of thin rods of flexible cellular cartilage, a condition thought to reflect the ancestral vertebrate state. To better understand the origin and evolution of the gnathostome head skeleton, we have been analyzing head skeleton development in the agnathan, lamprey. The fibroblast growth factors FGF3 and FGF8 have various roles during head development in jawed vertebrates, including pharyngeal pouch morphogenesis, patterning of the oral skeleton and chondrogenesis. We isolated lamprey homologs of FGF3, FGF8 and FGF receptors and asked whether these functions are ancestral features of vertebrate development or gnathostome novelties. Using gene expression and pharmacological agents, we found that proper formation of the lamprey head skeleton requires two phases of FGF signaling: an early phase during which FGFs drive pharyngeal pouch formation, and a later phase when they directly regulate skeletal differentiation and patterning. In the context of gene expression and functional studies in gnathostomes, our results suggest that these roles for FGFs arose in the first vertebrates and that the evolution of the jaw and gnathostome cellular cartilage was driven by changes developmentally downstream from pharyngeal FGF signaling.

The role of folate metabolism in orofacial development and clefting.

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

Lithium chloride promotes the odontoblast differentiation of hair follicle neural crest cells by activating Wnt/ß-catenin signaling.

The Wnt/ß-catenin signalling pathway contributes to the maintenance of pluripotency and partial reprogramming of stem cells. Postnatal neural crest cells (NCCs) can differentiate into odontoblast-like cells due to their multi-potential property, but further endeavors need to be made to promote odontogenic differentiation of hair follicle neural crest cells (hfNCCs). This study investigated whether the Wnt pathway activator lithium chloride (LiCl) promotes odontoblast differentiation of hfNCCs. Change of proliferation, ß-catenin and pluripotency markers of hfNCCs were examined after treatment with LiCl. An in vitro odontoblast differentiation model of hfNCCs was built using dental cell conditioned media (DC-CM). The effects of LiCl on odontoblast differentiation of hfNCCs showed that proliferation and expression of ß-catenin in the cytosolic and nuclear compartments were increased in the LiCl-treated hfNCCs, and the pluripotency marks, Oct4, Klf4, Sox2 and Nanog, were more highly expressed in the LiCl-treated group than in the control group. The odontoblast markers such as DSP, DMP1 and Runx2, could be detected in hfNCCs induced by DC-CM, but in LiCl -treated group all three markers had stronger expression. Expression of ß-catenin in the nuclear of LiCl-treated hfNCCs induced by DC-CM was higher than in the other groups. The data indicate that the Wnt pathway activator LiCl can promote proliferation and odontoblast differentiation of hfNCCs, and chemical approaches are of benefit in obtaining more desirable seed cell types for cell-based therapies.

Properties of neural crest-like cells differentiated from human embryonic stem cells.

Neural crest cells (NCCs) derive early in vertebrate ontogenesis from neural tube as a population of migratory cells with exquisite differentiation potential. Abnormalities in NCC behaviour are cause of debilitating diseases including cancers and a spectrum of neurocristopathies. Thanks to their multilineage differentiation capacity NCCs offer a cell source for regenerative medicine. Both these aspects make NCC biology an important issue to study, which can currently be addressed using methodologies based on pluripotent stem cells. Here we contributed to understanding the biology of human NCCs by refining the protocol for differentiation/propagation of NCClike cells from human embryonic stem cells and by characterizing the molecular and functional phenotype of such cells. Most importantly, we improved formulation of media for NCC culture, we found that poly-L-ornithine combined with fibronectin provide good support for NCC growth, we unravelled the tendency of cultured NCCs to maintain heterogeneity of CD271 expression, and we showed that NCCs derived here possess the capacity to react to BMP4 signals by dramatically up-regulating MSX1, which is linked to odontogenesis.

Altered gene expression in rat cranial neural crest cells exposed to a teratogenic glucose concentration in vitro: paradoxical downregulation of antioxidative defense genes.

BACKGROUND: Diabetic pregnancy is associated with increased risk of malformation in the infant. Diabetes-induced anomalies of the face and heart are strongly correlated with neural crest cell (NCC) maldevelopment. We aimed to study glucose-induced alterations of mRNA levels in cranial and trunk NCCs isolated from rat embryos with increased risk of developing mandibular and cardiac malformations in diabetic pregnancy. METHODS: Inbred Sprague-Dawley rat embryos were used for NCC isolation from neural tube explants. The migrating cells were exposed to 5.5 or 30 mmol/l glucose concentration for 48 hr, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (Dlx) or AP-2-α antibodies. RESULTS: Evaluation of the immunostained slides showed that approximately 75% of the cells were of NCC origin. Exposure to 30 mM glucose decreased mRNA levels of Copper-Zinc superoxide dismutase, manganese superoxide dismutase, extracellular superoxide dismutase, Catalase, Gpx-1, Nrf2, poly-ADP ribose polymerase, B-cell leukemia/lymphoma protein 2, and ß-Catenin genes in cranial neural crest explant cultures. In addition, Pax-3, Pax-6, Wnt3a, and Apc mRNA levels were decreased by high glucose exposure in both cranial and trunk neural crest explant cultures. CONCLUSION: Cranial NCCs diminish their mRNA levels of antioxidative enzymes and the Nrf2 response factor, as well as the antiapoptotic B-cell leukemia/lymphoma protein 2 gene, in response to increased ambient glucose concentration. Furthermore, both cranial and trunk NCC decrease the mRNA levels of the transcription factors Pax-3 and Pax-6, as well as key components of the Wnt pathway. These patterns of glucose-altered gene expression in a developmentally important cell population may be of etiological importance for NCC-associated malformations in diabetic pregnancy.

Irreversible effects of retinoic acid pulse on Xenopus jaw morphogenesis: new insight into cranial neural crest specification.

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.

Differentiation of Neural Crest Stem Cells in Response to Matrix Stiffness and TGF-ß1 in Vascular Regeneration.

The neural crest stem cells derived from human induced pluripotent stem cells (iPSC-NCSCs) are a valuable autologous cell source for tissue engineering and regenerative medicine. In this study, we investigated how iPSC-NCSCs could be regulated to regenerate arteries by microenvironmental factors, including the physical factor of matrix stiffness, and the chemical factor of transforming growth factor beta-1 (TGF-ß1). We found that, compared to soft substrate, stiff substrate drove iPSC-NCSCs differentiation into smooth muscle cells, which was further enhanced by TGF-ß1. To investigate the regulatory role of TGF-ß1 in vivo, we fabricated vascular grafts composed of electrospun nanofibrous scaffolds, collagen gel, iPSC-NCSCs, and TGF-ß1, and implanted them into athymic rats. The results showed that TGF-ß1 significantly promoted extracellular matrix synthesis and increased mechanical strength of vascular grafts. This study presents a proof of concept that iPSC-NCSCs can be used as a promising autologous cell source for vascular regeneration when combined with physical and chemical engineering.

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.

Developmental abnormalities of the otic capsule and inner ear following application of prolyl-hydroxylase inhibitors in chick embryos.

BACKGROUND: Naturally hypoxic conditions in amniote embryos play important roles in normal development. We previously showed that a hypoxic condition is required to produce a sufficient amount of neural crest cells (NCCs) during embryogenesis and that promoting a hypoxic response by prolyl-hydroxylase (PHD) inhibitors increases NCCs. Given that PHD inhibitors are considered as a potential treatment for anemia and ischemic diseases, we investigated the phenotypic effect of PHD inhibitors on embryonic development. METHODS: Chick embryos were administered with PHD inhibitors prior to the induction of NCCs on day 1.5. Three main events relating to hypoxia, NCCs induction, vasculogenesis and chondrogenesis, were examined. RESULTS: PHD inhibitors caused an increase of Sox10-positive NCCs in vivo. Vasculogenesis was promoted temporarily, although rapid vasculogenesis diminished the effect by day 5 in cephalic and pharyngeal regions. Studies on chondrogenesis at day 7 showed advanced development of the otic capsule, a cartilaginous structure encapsulating the inner ear. Analysis by X-ray micro-computed-tomography (µCT) revealed smaller otic capsule, suggesting premature differentiation. This in turn, deformed the developing semicircular canals within it. Other skeletal structures such as the palate and jaw were unaffected. The localized effect on the otic capsule was considered a result of the multiple effects from the hypoxic responses, increased NCCs and promoted chondrogenesis. CONCLUSION: Given the wide range of clinical applications being considered for PHD inhibitors, this study provides crucial information to caution and guide use of PHD inhibitors when treating women of childbearing age.

Differences in neural crest sensitivity to ethanol account for the infrequency of anterior segment defects in the eye compared with craniofacial anomalies in a zebrafish model of fetal alcohol syndrome.

BACKGROUND: Ethanol (ETOH) exposure during pregnancy is associated with craniofacial and neurologic abnormalities, but infrequently disrupts the anterior segment of the eye. In these studies, we used zebrafish to investigate differences in the teratogenic effect of ETOH on craniofacial, periocular, and ocular neural crest. METHODS: Zebrafish eye and neural crest development was analyzed by means of live imaging, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, immunostaining, detection of reactive oxygen species, and in situ hybridization. RESULTS: Our studies demonstrated that foxd3-positive neural crest cells in the periocular mesenchyme and developing eye were less sensitive to ETOH than sox10-positive craniofacial neural crest cells that form the pharyngeal arches and jaw. ETOH increased apoptosis in the retina, but did not affect survival of periocular and ocular neural crest cells. ETOH also did not increase reactive oxygen species within the eye. In contrast, ETOH increased ventral neural crest apoptosis and reactive oxygen species production in the facial mesenchyme. In the eye and craniofacial region, sod2 showed high levels of expression in the anterior segment and in the setting of Sod2 knockdown, low levels of ETOH decreased migration of foxd3-positive neural crest cells into the developing eye. However, ETOH had minimal effect on the periocular and ocular expression of transcription factors (pitx2 and foxc1) that regulate anterior segment development. CONCLUSION: Neural crest cells contributing to the anterior segment of the eye exhibit increased ability to withstand ETOH-induced oxidative stress and apoptosis. These studies explain the rarity of anterior segment dysgenesis despite the frequent craniofacial abnormalities in fetal alcohol syndrome. Birth Defects Research 109:1212-1227, 2017. © 2017 Wiley Periodicals, Inc.

[Retinoic acid signal pathway regulation of zebra fish tooth development through manipulation of the differentiation of neural crest].

OBJECTIVE: To investigate the mechanism of retinoic acid (RA) signal in dental evolution, RA is used to explore the influence of the mechanism on neural crest's migration during the early stage of zebra fish embryos. METHODS: We divided embryos of wild type and transgenic line zebra fish into three groups. 1 x 10(-7) to 6 x 10(-7) mol x L(-1) RA and 1 x 10(-7) mo x L(-1) 4-diethylaminobenzaldehyde (DEAB) were added into egg water at 24 hpf for 9 h. Dimethyl sulfoxid (DMSO) with the concentration was used as control group. Then, antisense probes of dlx2a, dlx2b, and barxl were formulated to perform whole-mount in situ hybridization to check the expressions of the genes in 48 hpf to 72 hpf embryos. We observed fluorescence of transgenic line in 4 dpf embryos. RESULTS: We obtained three mRNA probes successfully. Compared with DMSO control group, a low concentration (1 x 10(-7) mol x L(-1)) of RA could up-regulate the expression of mRNA (barx1, dlx2a) in neural crest. Obvious migration trend was observed toward the pharyngeal arch in which teeth adhered. Transgenic fish had spreading fluorescence tendency in pharyngeal arch. However, a high concentration (4 x 10(-7) mol x L(-1)) of RA malformed the embryos and killed them after treatment. One third of the embryos of middle concentration (3 x 10(-7) mo x L(-1)) exhibited delayed development. DEAB resulted in neural crest dysplasia. The expression of barxl and dlx2a were suppressed, and the appearance of dlx2b in tooth was delayed. CONCLUSION: RA signal pathway can regulate the progenitors of tooth by controlling the growth of the neural crest and manipulating tooth development

Effects of growth factors on the differentiation of neural crest cells and neural crest cell-derivatives.

During neurulation, neural crest cells migrate to many regions of the body to give rise to a wide variety of cell types. Many premigratory neural crest cells are pluripotent, their potency for differentiation being gradually restricted as they migrate along definite pathways and interact with factors present in the microenvironment. Effects of growth factors on these cells have been discussed in the present review. Mediation of growth factors in differentiation varies with the cell type. Growth factors exert a direct influence on the differentiation of neural and other related neural crest-derived tissues such as endocrinal tissues but evidence for such influences on neural crest-derived mesenchymal tissues is limited. For example, NGF, BDNF, and other factors present in neural tube extracts and glioma cell conditioned medium are essential for the differentiation of sensory neurons. Similarly, NGF, insulin, IGFs and possibly other undescribed factors are necessary for the differentiation of sympathetic neurons. IGFs also enhance the proliferation of mesenchymal derivatives of both neural crest and mesodermal origin. Glucocorticoid-mediated differentiation of neural crest-derived chromaffin endocrine cells that are ontogenetically closely related to sympathetic neurons can be inhibited by NGF, and chromaffin cells can be induced to express the neuronal phenotype by NGF. Some growth factors, such as NGF, act on neural crest- and not on placodally-derived neurons, whether the former are sensory or sympathetic. Placodal sensory neurons possess NGF receptors, but only display a limited response to NGF, perhaps because of low affinity of the receptors. Other growth factors, such as BDNF, selectively act upon sensory neurons, whether neural crest- or placodally-derived. Although extracellular matrix products play a role in initiating the differentiative process, signals from growth factors are necessary for the establishment of the functionally competent phenotype of neural crest-derived neurons, a situation that does not apply for neural crest-derived mesenchymal cells. It is interactions with ECM components deposited by epithelia that govern the differentiation of mesenchymal derivatives. Growth factors do effect proliferation of mesenchymal derivatives and inhibit mesenchymal differentiation. Although direct involvement of single growth factors in transformation o f one mesenchymal phenotype to another has not been reported so far, their localization at sites of epithelial-mesenchymal interactions in palate teeth and mandible, and the ability of excess growth factors to interrupt normal development is suggestive of their possible involvement. One group of growth factors, BMPs, can influence differentiation of cartilage, including those of neural crest origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural crest cell differentiation and carcinogenesis: capability of goldfish erythrophoroma cells for multiple differentiation and clonal polymorphism in their melanogenic variants.

Multiple differentiation shown by a single cell line (GEM 81) of goldfish erythrophoroma (tumors of integumental erythrophores) cells after administration of chemical induction in vitro includes 1) melanogenesis, 2) formation of reflecting platelets, 3) synthesis of pteridines heterogeneous to this species, 4) formation of dermal skeletons such as teeth and fin rays, 5) production of neuronal characters, and 6) genesis of lentoid bodies. Melanogenic cells, highest in inducibility, also show remarkable phenotypic diversification in their cell morphology, pigmentation, and physiologic response. In this paper, the following findings are presented; a) multiple differentiation shown by erythrophoroma cells occurs on a clonal basis, making whole component cells of a given induced colony strikingly similar in their cell characters, and b) induced melanogenic clones manifest a remarkable polymorphism in their melanosome ultrastructure and receptor composition associated with motile response. The divergence covers concentric lamellar, multivesicular, fibrillar, and macroglobular types for the former, and a varying combination of receptors for epinephrine, melanin concentrating hormone (MCH), and melatonin for the latter. Because a spectrum of phenotypes expressed by differentiation-induced erythrophoroma cells is restricted to those of neural crest origin (except lentoid bodies) and polymorphism in induced melanized cells is composed mostly of a collection of a variety of known melanogenic characters, it is presumed that erythrophoroma cells are capable of multiple differentiation within the commitment as neural crest cells.

Teratological research using in vitro systems. IV. Cells in culture.

Several in vitro cellular systems designed to screen agents for teratogenic potential are described in this report. These assays were selected from a review of literature published through the spring of 1986 that generated over 100 references on teratological research using cell-based systems. Some of the assays have a broader application than others, but most require confirmation by one or more additional complementary tests because of the specificity of the teratogenic mechanism the assays are investigating. Included are systems that use analysis of tumor cell attachment; intercellular communication; growth of human embryonic palatal mesenchyme cells; progesterone production in porcine granulosa cells; differentiation of embryonic neural crest, limb bud, midbrain, and Drosophila cells; and differentiation of tumor cells. Because of the dynamic nature of cell culture work, the group of assays listed here should not be viewed as encompassing all cell systems of value with regard to teratogenicity testing; instead, the list represents several of the more prominent systems now being evaluated by the scientific community.

Expression of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors in the mouse embryo.

Expression patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors during mouse embryogenesis were investigated using highly specific monoclonal antibodies. Differential and overlapping spatio-temporal patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor immunoreactivity were observed during active phases of morphogenesis of a variety of embryonic tissues, including neuroepithelia of brain and spinal cord, notochord, somites, cranial neural crest, craniofacial mesenchyme and epithelia, heart myocardium and endocardial cushions, tooth germs, whisker follicles, cartilage and striated muscle. The functional significance of these receptors was tested by exposing headfold stage mouse embryos to different subtype-selective 5-HT(2) receptor antagonists for 2 days in whole embryo culture. The most potent was the pan 5-HT(2) receptor antagonist ritanserin, which has high affinity for the 5-HT(2B) receptor. Ritanserin caused 100% malformed embryos at a dose of 1 microM. The 5-HT(2A/2C) receptor antagonist mianserin also caused a significant number of malformed embryos, but only when used at a 10 fold higher dose (10 microM). Ketanserin, which primarily targets 5-HT(2A) receptors, did not cause a significant number of malformed embryos at any dose tested. Together with previous evidence that 5-HT acts as an important morphoregulatory signal during mouse embryogenesis, present evidence for the early and continued expression of functional 5-HT(2) receptors throughout gestation raises the possibility that psychotropic drugs taken during pregnancy could interfere with developmental actions of 5-HT during prenatal development of neural and non-neural tissues.

Retinoic acid-induced asymmetric craniofacial growth and cleft palate in the TO mouse fetus.

The etiology and pathogenetic mechanisms of cleft palate (CP) are rather uncertain. Both genetic and environmental factors are known to cause failure of horizontalization and/or failure of fusion of the palatal shelves resulting in CP. Retinoic acid (RA)-induced CP in the mouse is reported to exhibit two peaks of incidence separated by a less sensitive window. The morphologic bases of the differential sensitivity are not known. The objectives of this study were to determine whether the TO mouse had similar peaks of sensitivity to RA-induced CP, and if it did, to evaluate the morphologic and histologic bases of CP induced at an early [Gestation Day (GD) 8] and at a late (GD 12) stage of embryonic development. Single doses of all-trans-RA were administered to groups of mice on one of GD 8 to 15. On GD 18, fetuses were evaluated for the presence of CP, and the developmental stage of the palatal shelves was determined. All doses of RA were found to induce a high incidence of CP in the GD 8 to 13 treatment groups. GD 14 and 15 were not susceptible. There were no stage-dependent peaks or less sensitive windows, indicating that RA-induced CP in this strain is a continuum from GD 8 through 13. Morphologically clefting in the GD 8-RA treatment group was characterized by extreme hypoplasia (65% to 100%, depending on the dose) or agenesis (35% in the 200 mg/kg group) of the palatal shelves and associated with astomia, microstomia, aglossia, microglossia, and micrognathia with fusion of mandible, maxilla, and zygoma. Treatment on subsequent days of gestation resulted in CP with the shelves reaching progressively higher levels of maturity in terms of developmental staging. There was no case of CP with horizontalized shelves apposing but failing to fuse with each other. The facial skeleton of GD 12-RA group was hypoplastic but not malformed. Reduction in all dimensions of the cranium and mandible was highly significant (P < 0.001) in the GD 8-RA group, whereas there was a clear imbalance between the vertical growth and that in other directions in the GD 12-RA group. The CR length, head and body weights, and the protein content of heads of GD 8-RA-treated embryos were significantly reduced. Histologic studies showed that both the intrinsic and extrinsic muscles of the tongue and face, growth of the Meckel's cartilage, and ossification of the mandible were severely affected in the GD 8 treatment group, whereas these tissues were only moderately affected in the embryos of the GD 12-RA group. However, the quality of cytodifferentiation of the muscles was not affected in either group. These data provide evidence for the susceptibility continuum of CP in this strain. They also indicate that agenesis and hypoplasia of the palatal shelves and primordia of craniofacial skeleton and musculature contribute to CP, the relative involvement of the components depending on the stage of drug administration. In the absence of pronounced cell death, it appears that RA possibly produces its deleterious effects on the precursors of craniofacial primordia, such as the neural crest, by misexpression of developmentally important genes.

Pathogenesis of retinoid-induced hindbrain malformations in an experimental model.

Among the findings associated with the human Retinoic Acid Embryopathy are hindbrain defects including the Arnold-Chiari malformation. The human Arnold-Chiari malformation (ACM) is a malformation complex where the cardinal feature is herniation of the caudal hindbrain into the vertebral column; it is frequently accompanied by lumbosacral myelorachischisis and hydrocephalus. Mice exposed to all-trans-retinoic acid or etretinate on day 8.25 of pregnancy, produce offspring with hindbrain herniation and caudal lumbosacral myelorachischisis in addition to a variety of other craniofacial and caudal malformations. Several experimental animals were observed to lack the caudal myelorachischisis proving that this lesion is not required to generate hindbrain herniation. We provide evidence that the cranial malformations, including hindbrain herniation, result from primary damage to the neural crest and the rhombencephalon. The vulnerability of these sites appears to be correlated with the presence of normal physiological cell death. While these experimental animals differ in many respects from the typical human Arnold-Chiari malformation, they may provide some insight into the pathogenesis of the latter.