Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure.

Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

Insights into stem cell factor chemotactic guidance of neural crest cells revealed by a real-time directionality-based assay.

The extracellular environment through which neural crest cells (NCCs) translocate and differentiate plays a crucial role in the determination of cell migration and homing. In the trunk, NCC-derived melanocyte precursor cells (MPCs) take the dorsolateral pathway and colonize the skin, where they differentiate into pigment cells (PCs). Our hypothesis was that the skin, the MPCs' target tissue, may induce a directional response of NCCs toward diffusible factor(s). We show that the treatment of in vitro NCCs with skin extract (SE) or Stem Cell Factor (SCF) contributes to maintaining proliferative activity, accelerates melanocyte differentiation, and guides a subpopulation of NCCs by chemotaxis toward the gradient source of these factors, suggesting that they may represent the MPCs' subpopulation. Current data on stimulated directional persistence of NCCs supports the participation of diffusible molecules in the target colonization mechanism, guiding MPCs to migrate and invade the skin. Our results show similar effects of SE and SCF on NCC growth, proliferation and pigment cell differentiation. Also, the use of a proven real-time directionality-based objective assay shows the directional migration of NCCs toward SE and SCF, indicating that the epidermal SCF molecule may be involved in the chemotactic guidance mechanism of in vivo NCCs. Although SCF is the strongest candidate to account for these phenomena, the nature of other factor(s) affecting NCC-oriented migration remains to be investigated. This data amplifies the functional scope of trophic factors by involving them in new cell behaviors such as molecular guidance in the colonization mechanism of embryonic cells.

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by neurotrophin 3.

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24-30h of incubation were treated during 10h with 100mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100mM ethanol for 10h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.

Ethanol induces morphological and dynamic changes on in vivo and in vitro neural crest cells.

BACKGROUND: Fetal alcohol syndrome (FAS) is an embryopathology related to maternal alcohol drinking. The information concerning the factors involved in the prenatal mechanisms of ethanol action at the cellular and molecular levels is scarce. Because several abnormal changes in FAS involve regions colonized by cell lineages derived from neural crest cells (NCCs), it is reasonable to propose that epigenetic alteration of this cell population can represent an important component of the etiopathogeny. The aim of this work was to evaluate the direct effect of ethanol on a chick embryo model, as well as on in vitro NCC morphology and dynamic behavior. METHODS: After ethanol treatment, in ovo or cultured chick embryos were used to determine the anatomical development of and to quantify the migratory parameters and apoptosis of NCCs. Scanning electron microscopy was performed on ethanol-perfused (and control) cultures of cephalic and trunk NCCs; the actin cytoskeleton was evaluated, and morphometric and dynamic parameters were determined after time-lapse videorecording. Recovery capacity after ethanol treatment was also determined. RESULTS: Chick embryos submitted to conditions sufficient to induce FAS in mammals displayed developmental disruptions frequently accompanied by cephalic/facial anomalies. In vitro studies also indicated that cephalic and trunk NCCs exposed to ethanol exhibited significant and permanent changes regarding cell shape, surface morphology, apoptotic cell death, cytoskeleton, and distance and velocity traveled, as well as an abnormal pattern of migration. CONCLUSIONS: Taking into account that even a limited period of abnormal behavior may imply serious consequences in the final cues of an embryonic cell population, our results indicate that the biological effects of ethanol on early development-even during a short time-could induce permanent ontogenetic perturbations of NCCs, with potentially dramatic effects on embryonic morphogenesis. These results support an important participation of NCCs in the etiopathogeny of FAS.