Embryonic exposure to the teratogen ethanol leads to dysmorphias, including eye and brain morphology defects associated with fetal alcohol spectrum disorder (FASD). Exposure of Xenopus laevis embryos to ethanol leads to similar developmental defects, including brain and eye dysmorphism, confirming our work and the work of others showing Xenopus as a useful system for studies of the brain and eye birth defects associated with FASD. Several targets of ethanol action have been hypothesized, one being regulation of Kir2.1 potassium channel. Endogenous ion fluxes and membrane voltage variation (bioelectric signals) have been shown to be powerful regulators of embryonic cell behaviors that are required for correct brain and eye morphology. Disruptions to these voltage patterns lead to spatially correlated disruptions in gene expression patterns and corresponding morphology.
Materials and Methods:
Here, we use controlled membrane voltage modulation to determine when and where voltage modulation is sufficient to rescue ethanol-induced brain and eye defects in Xenopus embryos.
Results:
We found (1) that modulating membrane voltage using light activation of the channelrhodopsin-2 variant D156A rescues ethanol exposed embryos, resulting in normal brain and eye morphologies; (2) hyperpolarization is required for the full duration of ethanol exposure; (3) hyperpolarization of only superficial ectoderm is sufficient for this effect; and(4) the rescue effect acts at a distance.
Conclusions:
These results, particularly the last, raise the exciting possibility of using bioelectric modulation to treat ethanol-induced brain and eye birth defects, possibly with extant ion channel drugs already prescribed to pregnant women. This may prove to be a simple and cost-effective strategy for reducing the impact of FASD.
Get full access to this article
View all access options for this article.
References
1.
CoppAJ, GreeneND. Genetics and development of neural tube defects. J Pathol, 2010; 220:217–230.
2.
ParriniE, ContiV, DobynsWB, et al.Genetic basis of brain malformations. Mol Syndromol, 2016; 7:220–233.
BatesEA.A potential molecular target for morphological defects of fetal alcohol syndrome: Kir2.1. Curr Opin Genet Dev, 2013; 23:324–329.
6.
FainsodA, Kot-LeibovichH. Xenopus embryos to study fetal alcohol syndrome, a model for environmental teratogenesis. Biochem Cell Biol, 2018; 96:77–87.
7.
CrewsFT, MorrowA.L, CriswellH, et al.Effects of ethanol on ion channels. Int Rev Neurobiol, 1996; 39:283–367.
8.
BukiyaAN, KuntamallappanavarG, EdwardsJ, et al. An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel. Proc Natl Acad Sci U S A, 2014; 111:9313–9318.
9.
OzM, TchugunovaY, DincM. Inhibition of cromakalim-activated K+ current by ethanol in follicle-enclosed Xenopus oocytes. Naunyn Schmiedebergs Arch Pharmacol, 2003; 367:80–85.
10.
ClaytonR, woodwardJJ. Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes. Neurochem Int, 1999; 36:67–74.
11.
MinamiK, GereauRWT, MinamiM, et al.Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes. Mol Pharmacol, 1998; 53:148–156.
12.
AoshimaH, HossainSJ, HamamotoK, et al.Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits. J Biochem, 2001; 130:703–709.
13.
SannaE, Dildy-MayfieldJE, HarrisRA. Ethanol inhibits the function of 5-hydroxytryptamine type 1c and muscarinic M1 G protein-linked receptors in Xenopus oocytes expressing brain mRNA: Role of protein kinase C. Mol Pharmacol, 1994; 45:1004–1012.
14.
ShukrunN, ShabtaiY, PillemerG, et al.Retinoic acid signaling reduction recapitulates the effects of alcohol on embryo size. Genesis, 2019; 57:e23284.
15.
ShabtaiY, FainsodA. Competition between ethanol clearance and retinoic acid biosynthesis in the induction of fetal alcohol syndrome. Biochem Cell Biol, 2018; 96:148–160.
16.
Kot-LeibovichH, FainsodA. Ethanol induces embryonic malformations by competing for retinaldehyde dehydrogenase activity during vertebrate gastrulation. Dis Model Mech, 2009; 2:295–305.
17.
PengY, YangPH, NgSS, et al.Protection of Xenopus laevis embryos against alcohol-induced delayed gut maturation and growth retardation by peroxiredoxin 5 and catalase. J Mol Biol, 2004; 340:819–827.
18.
PengY, YangPH, GuoY, et al.Catalase and peroxiredoxin 5 protect Xenopus embryos against alcohol-induced ocular anomalies. Invest Ophthalmol Vis Sci, 2004; 45:23–29.
19.
PengY, YangPH, NgSS, et al.A critical role of Pax6 in alcohol-induced fetal microcephaly. Neurobiol Dis, 2004; 16:370–376.
20.
ZhangY, WangH, LiY, et al.A review of interventions against fetal alcohol spectrum disorder targeting oxidative stress. Int J Dev Neurosci, 2018; 71:140–145.
21.
NuccitelliR.Endogenous electric fields in embryos during development, regeneration and wound healing. Radiat Prot Dosimetry, 2003; 106:375–383.
22.
JaffeLF.Control of development by steady ionic currents. Fed Proc, 1981; 40:125–127.
23.
McCaigCD, RajnicekAM, SongB, et al.Controlling cell behavior electrically: Current views and future potential. Physiol Rev, 2005; 85:943–978.
24.
AdamsDS, UzelSG, AkagiJ, et al.Bioelectric signalling via potassium channels: A mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome. J Physiol, 2016; 594:3245–3270.
25.
LevinM.Molecular bioelectricity in developmental biology: New tools and recent discoveries: Control of cell behavior and pattern formation by transmembrane potential gradients. Bioessays, 2012; 34:205–217.
26.
LevinM.Reprogramming cells and tissue patterning via bioelectrical pathways: Molecular mechanisms and biomedical opportunities. Wiley Interdiscip Rev Syst Biol Med, 2013; 5:657–676.
27.
PaiVP, MartyniukCJ, EcheverriK, et al.Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation. Regeneration (Oxf), 2016; 3:3–25.
28.
PaiVP, LevinM.Chapter 5: Bioelectric control of stem cell function. In: CalegariF, WaskovC, eds. Stem Cells: From Basic Research to Therapy. Vol 1. Chapter 5. Boca Raton, FL: CRC Press, 2014: 106–148.
29.
KimJB.Channelopathies. Korean J Pediatr, 2014; 57:1–18.
30.
AdamsDS, LevinM. General principles for measuring resting membrane potential and ion concentration using fluorescent bioelectricity reporters. Cold Spring Harb Protoc, 2012; 2012:385–397.
31.
VandenbergLN, MorrieRD, AdamsDS. V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis. Dev Dyn, 2011; 240:1889–1904.
32.
PaiVP, AwS, ShomratT, et al.Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis. Development, 2012; 139:313–323.
33.
PaiVP, LemireJM, ChenY, et al.Local and long-range endogenous resting potential gradients antagonistically regulate apoptosis and proliferation in the embryonic CNS. Int J Dev Biol, 2015; 59:327–340.
34.
PaiVP, LemireJM, PareJF, et al.Levin Endogenous gradients of resting potential instructively pattern embryonic neural tissue via Notch signaling and regulation of proliferation. J Neurosci, 2015; 35:4366–4385.
35.
PaiVP, VandenbergLN, BlackistonD, et al.Neurally derived tissues in Xenopus laevis embryos exhibit a consistent bioelectrical left-right asymmetry. Stem Cells Int, 2012; 2012:353491.
36.
BronnerME.Formation and migration of neural crest cells in the vertebrate embryo. Histochem Cell Biol, 2012; 138:179–186.
37.
BronnerME, Simões-CostaM. The neural crest migrating into the twenty-first century. Curr Top Dev Biol, 2016; 116:115–134.
38.
StoneLS.Experiments on the transplantation of placodes of the cranial ganglia in the amphibian embryo I. Heterotopic transplantations of the ophthalmic placode upon the head of Amblystoma punctatum. J Comp Neurol, 1924; 38:73–105.
39.
AdameykoI, FriedK. The nervous system orchestrates and integrates craniofacial development: A review. Front Physiol, 2016; 7:49.
40.
MoodySA, PetersonMC, XuJN, et al.The molecular regulation of cranial placode specification. FASEB J, 2011; 25:184.1.
41.
SchlosserG, AwtryT, BrugmannSA, et al.Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion. Dev Biol, 2008; 320:199–214.
42.
PaiVP, PietakA, WillocqV, et al.HCN2 Rescues brain defects by enforcing endogenous voltage pre-patterns. Nat Commun, 2018; 9:998.
43.
Herrera-RinconC, PaiVP, MoranKM, et al.The brain is required for normal muscle and nerve patterning during early Xenopus development. Nat Commun, 2017; 8:587.
44.
FennoL, YizharO, DeisserothK. The development and application of optogenetics. Annu Rev Neurosci, 2011; 34:389–412.
45.
KnopfelT, LinMZ, LevskayaA, et al.Toward the second generation of optogenetic tools. J Neurosci, 2010; 30:14998–15004.
AdamsDS, TsengAS, LevinM. Light-activation of the Archaerhodopsin H(+)-pump reverses age-dependent loss of vertebrate regeneration: Sparking system-level controls in vivo. Biol Open, 2013; 2:306–313.
48.
ChernetBT, AdamsDS, LobikinM, et al.Use of genetically encoded, light-gated ion translocators to control tumorigenesis. Oncotarget, 2016; 7:19575–19588.
49.
NakatsujiN.Craniofacial malformation in Xenopus laevis tadpoles caused by the exposure of early embryos to ethanol. Teratology, 1983; 28:299–305.
50.
YelinR, KotH, YelinD, et al.Early molecular effects of ethanol during vertebrate embryogenesis. Differentiation, 2007; 75:393–403.
51.
SiveH, GraingerRM, HarlandR.Early Development of Xenopus laevis. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2000: 338.
52.
NieuwkoopPD, FaberJ. Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the end of Metamorphosis. New York: Garland Pub, 1994: 252.
53.
ZahnN, LevinM, AdamsDS. The Zahn drawings: New illustrations of Xenopus embryo and tadpole stages for studies of craniofacial development. Development, 2017; 144:2708–2713.
54.
PrattKG, KhakhalinAS. Modeling human neurodevelopmental disorders in the Xenopus tadpole: From mechanisms to therapeutic targets. Dis Model Mech, 2013; 6:1057–1065.
55.
BamannC, GuetaR, KleinlogelS, et al.Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond. Biochemistry, 2010; 49:267–278.
56.
Lorenz-FonfriaVA, HeberleJ. Channelrhodopsin unchained: Structure and mechanism of a light-gated cation channel. Biochim Biophys Acta, 2014; 1837:626–642.
57.
AdamsDS, LemireJM, KramerRH, et al.Optogenetics in developmental biology: Using light to control ion flux-dependent signals in Xenopus embryos. Int J Dev Biol, 2014; 58:851–861.
58.
LinJY, LinMZ, SteinbachP, et al.Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J, 2009; 96:1803–1814.
59.
MoodySA.Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev Biol, 1987; 122:300–319.
60.
AdamsDS, RobinsonKR, FukumotoT, et al.Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. Development, 2006; 133:1657–1671.
61.
AwS, AdamsDS, QiuD, et al.H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry. Mech Dev, 2008; 125:353–372.
62.
AdamsDS, LevinM. Endogenous voltage gradients as mediators of cell-cell communication: Strategies for investigating bioelectrical signals during pattern formation. Cell Tissue Res, 2013; 352:95–122.
63.
LevinM.Endogenous bioelectrical networks store non-genetic patterning information during development and regeneration. J Physiol, 2014; 592(Pt 11):2295–2305.
64.
TsengA, LevinM. Cracking the bioelectric code: Probing endogenous ionic controls of pattern formation. Commun Integr Biol, 2013; 6:e22595.
65.
MathewsJ, LevinM. The body electric 2.0: Recent advances in developmental bioelectricity for regenerative and synthetic bioengineering. Curr Opin Biotechnol, 2018; 52:134–144.
PennAA, ShatzCJ. Brain waves and brain wiring: The role of endogenous and sensory-driven neural activity in development. Pediatr Res, 1999; 45(4 Pt 1):447–458.
68.
BeaneWS, MorokumaJ, LemireJM, et al.Bioelectric signaling regulates head and organ size during planarian regeneration. Development, 2013; 140:313–322.
69.
HempelA, KuhlM. A matter of the heart: The African clawed frog xenopus as a model for studying vertebrate cardiogenesis and congenital heart defects. J Cardiovasc Dev Dis, 2016; 3.
70.
PopovS, VenetsanouK, ChedresePJ, et al.Bertorello Increases in intracellular sodium activate transcription and gene expression via the salt-inducible kinase 1 network in an atrial myocyte cell line. Am J Physiol Heart Circ Physiol, 2012; 303:H57–H65.
71.
HuangBS, WhiteRA, LeenenFH. Possible role of brain salt-inducible kinase 1 in responses to central sodium in Dahl rats. Am J Physiol Regul Integr Comp Physiol, 2012; 303:R236–R245.
72.
AllahverdiA, ChenQ, KorolevN, et al.Chromatin compaction under mixed salt conditions: Opposite effects of sodium and potassium ions on nucleosome array folding. Sci Rep, 2015; 5:8512.
73.
LinJY, LinMZ, SteinbachP, et al.Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J, 2009; 96:1803–1814.
74.
YelinR, SchyrRB, KotH, et al.Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels. Dev Biol, 2005; 279:193–204.
75.
EomDS, ParichyDM. A macrophage relay for long-distance signaling during postembryonic tissue remodeling. Science, 2017; 355:1317–1320.
76.
MondiaJP, LevinM, OmenettoFG, et al.Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation. PLoS One, 2011; 6:e24953.
77.
KumarA, BrockesJP. Nerve dependence in tissue, organ, and appendage regeneration. Trends Neurosci, 2012; 35:691–699.
78.
Rosello-DiezA, MadisenL, BastideS, et al.Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice. PLoS Biol, 2018; 16:e2005086.
79.
MarburgerRG, RobinsonRM, ThomasJW, et al.Antler malformation produced by leg injury in white-tailed deer. J Wildl Dis, 1972; 8:311–314.
80.
AcharjyoLN, MisraR. Effects of amputation of a hind limb on the growth of antlers of deer in captivity. Indian Forester, 1972; 98:507–508.
81.
OviedoNJ, MorokumaJ, WalentekP, et al.Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration. Dev Biol, 2010; 339:188–199.
82.
ChernetBT, FieldsC, LevinM. Long-range gap junctional signaling controls oncogene-mediated tumorigenesis in Xenopus laevis embryos. Front Physiol, 2014; 5:519.
83.
LobikinM, ChernetB, LoboD, et al.Resting potential, oncogene-induced tumorigenesis, and metastasis: The bioelectric basis of cancer in vivo. Phys Biol, 2012; 9:065002.
84.
LevineE, LeeCH, KintnerC, et al.Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant. Development, 1994; 120:901–909.
85.
NandadasaS, TaoQ, MenonNR, et al.N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements. Development, 2009; 136:1327–1338.
86.
GoldsmithDR, WagstaffAJ, IbbotsonT, et al.Lamotrigine: A review of its use in bipolar disorder. Drugs, 2003; 63:2029–2050.
87.
DolkH, WangH, LoaneM, et al.Lamotrigine use in pregnancy and risk of orofacial cleft and other congenital anomalies. Neurology, 2016; 86:1716–1725.
88.
WiffenPJ, DerryS, BellRF, et al.Gabapentin for chronic neuropathic pain in adults. Cochrane Database Syst Rev, 2017; 6:CD007938.
