Neural Crest Apoptosis Research Articles

Alcohol induces p53-mediated apoptosis in neural crest by stimulating an AMPK-mediated suppression of TORC1, S6K, and ribosomal biogenesis

Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability and can feature distinctive craniofacial deficits that partly originate from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of ribosome biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol’s activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. Alcohol rapidly (<2hr) increased pAMPK, pTSC2, and pRaptor, and reduced both total and pS6K in NC cells. These changes persisted for at least 12hr to 18hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches that targeted AMPK, S6K, or TORC1 prevented alcohol’s suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates TSC2 and Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol’s neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.

Alcohol induces p53-mediated apoptosis in neural crest by stimulating an AMPK-mediated suppression of TORC1, S6K, and ribosomal biogenesis.

Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability and can feature distinctive craniofacial deficits that partly originate from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of ribosome biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol's activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. Alcohol rapidly (<2hr) increased pAMPK, pTSC2, and pRaptor, and reduced both total and pS6K in NC cells. These changes persisted for at least 12hr to 18hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches that targeted AMPK, S6K, or TORC1 prevented alcohol's suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates TSC2 and Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol's neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.

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.

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. 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. 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. 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.

Editorial: Genetics and epigenetics of fetal alcohol spectrum disorders.

Editorial: Genetics and epigenetics of fetal alcohol spectrum disorders.

High-throughput transcriptome sequencing identifies candidate genetic modifiers of vulnerability to fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized. We performed high-throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanol's (EtOH's) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well-characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/β-catenin-dependent apoptosis. Samples were not exposed to EtOH prior to analysis. We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10(-47) ). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10(-11) ), spliceosome (13 genes, p = 7.02 × 10(-8) ), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/β-catenin signals that mediate EtOH's neurotoxicity in this model, including β-catenin itself and both calmodulin isoforms. Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53-mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH-treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approach's efficacy to formulate new, mechanistic hypotheses regarding EtOH's developmental damage.

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.

Murine craniofacial development requires Hdac3-mediated repression of Msx gene expression

Craniofacial development is characterized by reciprocal interactions between neural crest cells and neighboring cell populations of ectodermal, endodermal and mesodermal origin. Various genetic pathways play critical roles in coordinating the development of cranial structures by modulating the growth, survival and differentiation of neural crest cells. However, the regulation of these pathways, particularly at the epigenomic level, remains poorly understood. Using murine genetics, we show that neural crest cells exhibit a requirement for the class I histone deacetylase Hdac3 during craniofacial development. Mice in which Hdac3 has been conditionally deleted in neural crest demonstrate fully penetrant craniofacial abnormalities, including microcephaly, cleft secondary palate and dental hypoplasia. Consistent with these abnormalities, we observe dysregulation of cell cycle genes and increased apoptosis in neural crest structures in mutant embryos. Known regulators of cell cycle progression and apoptosis in neural crest, including Msx1, Msx2 and Bmp4, are upregulated in Hdac3-deficient cranial mesenchyme. These results suggest that Hdac3 serves as a critical regulator of craniofacial morphogenesis, in part by repressing core apoptotic pathways in cranial neural crest cells.

Calcium-mediated repression of β-catenin and its transcriptional signaling mediates neural crest cell death in an avian model of fetal alcohol syndrome

Fetal alcohol syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates G-protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol's calcium transient and effect neural crest death are unknown. Here we show that ethanol's repression of the transcriptional effector β-catenin causes the neural crest losses. Clinically relevant ethanol concentrations (22-78 mM) rapidly deplete nuclear β-catenin from neural crest progenitors, with accompanying losses of β-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion, and survival. Using forced expression studies, we show that β-catenin loss of function (via dominant-negative T cell transcription factor [TCF]) recapitulates ethanol's effects on neural crest apoptosis, whereas β-catenin gain-of-function in ethanol's presence preserves neural crest survival. Blockade of ethanol's calcium transient using Bapta-AM normalizes β-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize β-catenin. We propose that ethanol's repression of β-catenin causes the neural crest losses in this model of FAS. β-Catenin is a novel target for ethanol's teratogenicity. β-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.

CaMKII activation is a novel effector of alcohol’s neurotoxicity in neural crest stem/progenitor cells

Prenatal ethanol exposure causes significant neurodevelopmental deficits through its induction of apoptosis in neuronal progenitors including the neural crest. Using an established chick embryo model, we previously showed that clinically relevant ethanol concentrations cause neural crest apoptosis through mobilization of an intracellular calcium transient. How the calcium transient initiates this cell death is unknown. In this study, we identify CaMKII as the calcium target responsible for ethanol-induced apoptosis. Immunostaining revealed selective enrichment of activated phosphoCaMKII(Thr286) within ethanol-treated neural crest. CaMKII activation in response to ethanol was rapid (< 60 s) and robust, and CaMKII activity was increased 300% over control levels. Treatment with CaMKII-selective inhibitors but not those directed against CaMKIV or PKC completely prevented the cell death. Forced expression of dominant-negative CaMKII prevented ethanol's activation of CaMKII and prevented the ethanol-induced death, whereas constitutively active CaMKII in ethanol's absence significantly increased cell death to levels caused by ethanol treatment. In summary, CaMKII is the key signal that converts the ethanol-induced, short-lived Ca(i) (2+) transient into a long-lived cellular effector. This is the first identification of CaMKII as a critical mediator of ethanol-induced cell death. Because neural crest differentiates into several neuronal lineages, our findings offer novel insights into how ethanol disrupts early neurogenesis.

Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in Patch embryos

Although Patch mutants show severe abnormalities in many neural crest-derived structures including the face and the heart, there is a paucity of information characterizing the mechanisms underlying these congenital defects. Via manipulating the genetic background to circumvent early embryonic lethality, our results revealed that Patch phenotypes are most likely due to a significant decrease in migratory neural crest lineage due to diminished neural crest survival and elevated apoptosis. Homozygous mutant neural crest precursors can undergo typical expansion within the neural tube, epithelial-to-mesenchymal transformation, and initiate normal neural crest emigration. Moreover, in vitro explant culture demonstrated that when isolated from the surrounding mesenchyme, Patch mutant neural crest cells (NCCs) can migrate appropriately. Additionally, Patch foregut, notochord and somitic morphogenesis, and Sonic hedgehog expression profiles were all perturbed. Significantly, the timing of lethality and extent of apoptosis correlated with the degree of severity of Patch mutant foregut, notochord, and somite dysfunction. Finally, analysis of Balb/c-enriched surviving Patch mutants revealed that not all the neural crest subpopulations are affected and that Patch mutant neural crest-derived sympathetic ganglia and dorsal root ganglia were unaffected. We hypothesize that loss of normal coordinated signaling from the notochord, foregut, and somites underlies the diminished survival of the neural crest lineage within Patch mutants resulting in subsequent neural crest-deficient phenotypes.

α5β1 Integrin Blockade Inhibits Lymphangiogenesis in Airway Inflammation

The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.

Ethanol Triggers Neural Crest Apoptosis through the Selective Activation of a Pertussis Toxin–Sensitive G Protein and a Phospholipase Cβ–Dependent Ca2+ Transient

Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient. Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes. Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells. We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.

Ethanol-induced cephalic apoptosis requires phospholipase C-dependent intracellular calcium signaling.

Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis. Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange. Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis. Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.

Genetic Influences on Craniofacial Outcome in an Avian Model of Prenatal Alcohol Exposure

Background: Understanding the basis for ethanol's teratogenic effects may inform the etiology of fetal alcohol syndrome. Here we investigate how genetic background and susceptibility to ethanol-induced neural crest apoptosis contribute to the distinctive craniofacial phenotype observed after prenatal alcohol exposure. Methods: Nine different chick strains were exposed to ethanol at gastrulation. The sensitivity of these embryos to ethanol-induced neural crest apoptosis was reported elsewhere (Debelak and Smith, 2000). Here, these embryos were permitted to develop until embryonic day 10, when facial morphogenesis was largely complete, and cephalometric measurements were made on cleared skulls. Shifts in facial growth were correlated against the severity of ethanol-induced apoptosis in facial precursors. Results: The facial shape produced by ethanol exposure was a function of the embryos’ genetic strain. Three general responses were observed: apparent midfacial flattening (Babcock B300 x Hampshire Red, ISA-Babcock, HyLine W98, and HyLine W36 strains), overall facial expansion (Spafas and Babcock B300 strains), or overall facial hypoplasia (DeKalb strains). When dose and timing of exposure were held constant, the embryo's genetic background predicted the facial outcome. For ethanol-sensitive strains, apoptosis of facial precursor populations was required to produce the facial defects. That some strains had essentially normal faces despite extensive cell death indicated a capacity to recover from the earlier neural crest losses. Conclusions: We propose that ethanol's effects on craniofacial development are multifactoral, and these influences may include susceptibility to apoptosis, regenerative capacity, and compensatory outgrowth of the facial primordia. The embryo's genetic background may modulate these events. The high and low responder chick strains are useful tools to dissect these contributions.

Genetic Influences on Craniofacial Outcome in an Avian Model of Prenatal Alcohol Exposure

Understanding the basis for ethanol's teratogenic effects may inform the etiology of fetal alcohol syndrome. Here we investigate how genetic background and susceptibility to ethanol-induced neural crest apoptosis contribute to the distinctive craniofacial phenotype observed after prenatal alcohol exposure. Nine different chick strains were exposed to ethanol at gastrulation. The sensitivity of these embryos to ethanol-induced neural crest apoptosis was reported elsewhere (Debelak and Smith, 2000). Here, these embryos were permitted to develop until embryonic day 10, when facial morphogenesis was largely complete, and cephalometric measurements were made on cleared skulls. Shifts in facial growth were correlated against the severity of ethanol-induced apoptosis in facial precursors. The facial shape produced by ethanol exposure was a function of the embryos' genetic strain. Three general responses were observed: apparent midfacial flattening (Babcock B300 x Hampshire Red, ISA-Babcock, HyLine W98, and HyLine W36 strains), overall facial expansion (Spafas and Babcock B300 strains), or overall facial hypoplasia (DeKalb strains). When dose and timing of exposure were held constant, the embryo's genetic background predicted the facial outcome. For ethanol-sensitive strains, apoptosis of facial precursor populations was required to produce the facial defects. That some strains had essentially normal faces despite extensive cell death indicated a capacity to recover from the earlier neural crest losses. We propose that ethanol's effects on craniofacial development are multifactoral, and these influences may include susceptibility to apoptosis, regenerative capacity, and compensatory outgrowth of the facial primordia. The embryo's genetic background may modulate these events. The high and low responder chick strains are useful tools to dissect these contributions.

Avian Genetic Background Modulates the Neural Crest Apoptosis Induced by Ethanol Exposure

Ethanol-induced neural crest apoptosis likely contributes to the distinctive craniofacial phenotype that results from prenatal alcohol exposure. The mechanism responsible for this apoptosis is incompletely understood. A serendipitous change in poultry production flocks led to the discovery that, in chick, the embryo's genetic background modulates its susceptibility to ethanol-induced apoptosis. We examined the level of ethanol-induced neural crest apoptosis in 11 chick layer strains or crosses, using acridine orange uptake. Holding the ethanol dose and exposure stage constant, strains were classified into very sensitive (Babcock ISA, HyLine W98, Babcock B300/Hampshire Red cross [BxHR]), moderately sensitive (Spafas, HyLine W36, Babcock B300), and nonresponsive (DeKalb White and Black, Shaver White and 2000, DcKalb White/Hampshire Red cross). Detailed examination of two susceptible strains (W98, BxHR) and a resistant strain (DcKalb White) revealed that the DeKalb's nonresponse was not caused by a shift in timing of apoptosis, or to a lower alcohol exposure at either time of injection or time of death. Strains had identical stage distributions at the time of injection and at apoptosis; housing and diet were held constant. Factors within the embryo and/or egg environment can affect the susceptibility to ethanol-induced apoptosis. These sensitive and resistant strains will be important tools to dissect the molecular mechanism of ethanol-induced apoptosis, and for understanding how these losses affect subsequent development.

Avian Genetic Background Modulates the Neural Crest Apoptosis Induced by Ethanol Exposure

Avian Genetic Background Modulates the Neural Crest Apoptosis Induced by Ethanol Exposure

Ethanol‐Induced Neural Crest Apoptosis Is Coincident with Their Endogenous Death, But Is Mechanistically Distinct

The ability of both acute and chronic ethanol exposures to elicit cell death within specific embryonic and adult tissues is believed to partly underlie ethanol's pathogenicity; however, the mechanism underlying this cell death is unknown. This study partially characterized the mechanism of ethanol-induced neural crest cell death in a chick embryo model of fetal alcohol syndrome. In situ DNA end-labeling demonstrated this cell death was apoptotic and occurred at embryonic ethanol levels as low as 42 mM. Regardless of the initial exposure time, this apoptosis always appeared at a distinct developmental time point simultaneous with the normal deletion of a cranial neural crest subset. This suggested that ethanol might act through aberrant activation of the endogenous death pathway; however, ethanol exposure failed to induce two components of this pathway, the homeotic transcription factor msx-2 and the growth factor bone morphogenetic protein 4. Both endogenous and ethanol-induced death were blocked by local application of an interleukin-1beta converting enzyme/CED-3 protease (caspase) inhibitor, showing that the two paths converge mechanistically and suggesting the potential to prevent this aspect of ethanol's teratogenicity. Ethanol exposure did not significantly alter cell proliferation within neural crest-populated regions, suggesting that susceptibility to ethanol-induced death did not involve exit from the cell cycle. Apoptotic deletion of cranial neural crest could partially explain the craniofacial deficits characteristic of the fetal alcohol syndrome.

Ethanol-Induced Neural Crest Apoptosis Is Coincident with Their Endogenous Death, But Is Mechanistically Distinct

Ethanol-Induced Neural Crest Apoptosis Is Coincident with Their Endogenous Death, But Is Mechanistically Distinct

Mesodermal defects and cranial neural crest apoptosis in alpha5 integrin-null embryos.

Alpha5beta1 integrin is a cell surface receptor that mediates cell-extracellular matrix adhesions by interacting with fibronectin. Alpha5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the alpha5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of alpha5-null embryos compared to wild-type controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in the alpha5-null embryos. No increases in cell death were seen in alpha5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in alpha5-null embryos, indicating that these subpopulations of cells are dependent on alpha5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh) were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of alpha5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the alpha5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the alpha5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in alpha5-null embryos. These results suggest that alpha5beta1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of mesodermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.