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, 91 (7), 591-602

Calcium-mediated Repression of β-Catenin and Its Transcriptional Signaling Mediates Neural Crest Cell Death in an Avian Model of Fetal Alcohol Syndrome

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Calcium-mediated Repression of β-Catenin and Its Transcriptional Signaling Mediates Neural Crest Cell Death in an Avian Model of Fetal Alcohol Syndrome

George R Flentke et al. Birth Defects Res A Clin Mol Teratol.

Abstract

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.

Figures

Figure 1
Figure 1
Ethanol exposure causes neural crest cell death. (A) Diagram of 3-somite embryo showing boxed headfold region imaged for intracellular calcium. (B, C) Fura-2 imaging of intracellular calcium in a 3-somite embryo following challenge with saline (B) and 52 mM ethanol (C). Asterisk indicates dorsal neural fold enriched in neural crest progenitors. (D, E) Cell death is visualized in 17-somite embryos using LysoTracker Red (LTR, white dots). (D) Saline (Sal) control reveals programmed cell death at the dorsal midline of the midbrain (mb) and hindbrain rhombomeres r1/2, r3 and r5 (arrows). (E) Ethanol treatment (52 mM) substantially increases the number of LTR+ cells within the midbrain and hindbrain (compare signal at arrows). (F, G) Transverse histological sections through rhombomere 4 of 17-somite embryos immunostained for Slug and cell death (LTR). Dorsal is at the top. Saline-treated r4 (F) contains a substantial slug+ neural crest population (green signal at arrow) with few apoptotic neural crest (red signal). In contrast, the ethanol-treated r4 (G) has many apoptotic neural crest cells, shown in yellow (green-red overlay, arrows). OV, otic vesicle.
Figure 2
Figure 2
Acute ethanol treatment rapidly depletes transcriptionally active β-catenin from neural crest. (A, B) Transverse sections through presumptive hindbrain of 3-somite embryo treated 2hr ± 52 mM ethanol, dorsal at top, and stained for β-catenin protein (green) and nuclei (blue). Control hindbrain (A) shows strong β-catenin expression within dorsal regions including dorsolateral populations enriched in neural crest progenitors (boxed region). In contrast, ethanol-treated hindbrain (B) has significant β-catenin loss in the nuclei of dorsal populations including premigratory neural crest (arrows). Insets show magnified views of boxed regions; arrows indicate nuclear signal. m, midline; nc, neural crest; nf, neural folds. Scale bar equals 50 μm. Exposure time was constant between images. (C) Western blot analysis confirms the dose-dependent loss of β-catenin content following 2hr ethanol exposure. β-catenin content is normalized to GAPDH. One-way ANOVA with Holm-Sidak post-hoc analysis, * p < 0.01, ** p ≤ 0.001 vs. 0 mM ethanol. Mean ± SEM for 3–5 independent experiments. (D) Representative western blot shows the decline in β-catenin content with increasing ethanol concentration. Because each lane contains the total cranial protein from a single embryo, there is loading variation between lanes that is normalized against GAPDH content. Upper arrow, intact β-catenin; lower arrow, proteolytic fragments of β-catenin. (E) Western blot showing the increased phospho-β-catenin content and lower molecular weight protein fragments following 2hr challenge with 52 mM ethanol.
Figure 3
Figure 3
Ethanol significantly reduced β-catenin/TCF transcriptional activity. (A) β-Catenin/Tcf transcriptional activity measured using the pBARL luciferase reporter. Left side: Significant decreases were found at all ethanol concentrations tested. Middle: No luciferase activity was obtained using the pfuBARL reporter that contains mutant Tcf binding sequences. Right side: Recombinant luciferase and Renilla luciferase were not inhibited by 52 mM ethanol. Kruskal-Wallis one-way ANOVA on ranks with Dunn’s post hoc analysis, * p<0.05 vs. 0 mmol ethanol. Mean ± SEM of 4–6 experiments with 6–8 dissected crania/treatment analyzed individually. (B) Treatment with 52 mM ethanol does not alter short-term expression of canonical Wnt effectors in neural crest, as quantified using real-time PCR. Within-treatment values were normalized against GAPDH expression. Transcript levels in ethanol-treated neural crest were then normalized against the saline-treated levels. Shown is mean ± SD of 3–5 experiments using 25–30 pooled HH8/9 crania assayed in triplicate.
Figure 4
Figure 4
Ethanol selectively represses transcriptional targets of β-catenin/TCF signaling in neural crest. (A) Transcript levels in HH9 neural crest 6hr after ethanol (52 mM) challenge, normalized to saline controls (100%). Ethanol selectively reduced the expression of β-catenin/TCF-dependent neural crest genes Wnt6, FoxD3 and Slug, but not the neural crest genes Tgfβ3, Bmp4 or Snail. Mean ± SD of 5–6 pooled crania assayed in triplicate. * p <0.05 using unpaired t-test. (B) Time course analysis revealed that ethanol’s reduction of FoxD3 (⋄) and Wnt6 (Δ) was developmentally persistent and the reduction in slug (○) was transient. Expression of the β-catenin-independent genes Snail (▲), Tgfβ2 (■) and Bmp4 (◆) were largely unaffected until the onset of neural crest apoptosis (stage 12/13). Shown is mean expression with SEMs omitted for clarity; these averaged 10–15% of mean values. Values were normalized to GAPDH and expressed relative to saline controls (100%).
Figure 5
Figure 5
β-catenin/TCF misexpression alters ethanol-induced cell death and neural crest survival. (A) β-catenin/TCF transcriptional activity in neural crest expressing β-catenin/TCF constructs. Ethanol treatment significantly reduces β-catenin/TCF transcriptional activity and this is normalized by forced expression of β-catenin, whereas dominant-negative ΔTCF inhibits β-catenin/TCF activity in both treatment groups. Activities were normalized to eGFP-saline controls. Mean ± SEM of 3–4 experiments. *p<0.05 vs. saline-eGFP. (B) Quantitation of LTR+-slug+ neural crest following the indicated treatments. β-catenin significantly reduced the number of apoptotic neural crest in ethanol treated hindbrain, whereas ΔTCF increased neural crest death to levels caused by ethanol. Mean ± SEM of 3–4 embryos per group. *p<0.05 vs. Saline-eGFP; † p<0.05 vs. Ethanol-eGFP. (C) Quantitation of slug+ neural crest cells following the indicated treatments. Forced expression of β-catenin significantly increased the number of slug+ neural crest in both ethanol and saline-treated hindbrain, whereas ΔTCF lowered those numbers to that caused by ethanol. Mean ± SEM of 3–4 embryos per group. * p<0.025 vs. Saline-eGFP; † p=0.006 vs. Ethanol-eGFP. (A, B) were analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s post hoc analysis. (C) was analyzed using one-way ANOVA and Student-Newman-Kuels post-hoc analysis
Figure 6
Figure 6
Forced expression of β-catenin and ΔTCF alters ethanol-induced cell death and neural crest numbers. Shown are transverse sections of the r4 dorsal roof, using representative embryos analyzed in Figure 4. Arrows highlight individual slug+ cells (green). (A, B) Ethanol (B) appreciably increased the number of apoptotic slug+ neural crest as compared with saline controls (A). (C, D) Forced expression of β-catenin increased the number of slug+ neural crest and decreased the number of apoptotic slug+ cells, following saline (C) and ethanol treatment (D). (E, F) In contrast, ΔTCF substantially decreased the number of slug+ cells and increased the number of apoptotic cells within r4 following both saline (E) and ethanol treatment (F).
Figure 7
Figure 7
Neural fold β-catenin content is altered by ethanol-induced Cai+2 signals. Embryos having 3 somites were challenged with saline, ethanol and/or the indicated calcium effector, and β-catenin protein (green, arrows) was visualized 2hr thereafter. Righthand panels show a 40× magnification of each right dorsal neural fold where premigratory neural crest resides. (A) Saline-treated embryo has robust β-catenin distribution in the dorsal neural folds (arrows). (B) 52 mM ethanol substantially reduces β-catenin content in the dorsal neural folds. (C) Embryo pretreatment with the Cai+2 chelator Bapta-AM prior to 52 mM ethanol challenge prevents β-catenin loss. (D) Treatment of otherwise normal embryos with the calcium ionophore Ionomycin is sufficient to deplete β-catenin from dorsal neural folds.
Figure 8
Figure 8
Cai+2 effectors modulate β-catenin transcriptional activity and neural crest survival. (A) β-catenin/TCF transcriptional activity in neural crest treated with Bapta-AM ± 52 mM ethanol, measured using embryonic crania transfected with the pBARL luciferase reporter. Bapta pretreatment reverses the ethanol-mediated suppression of β-catenin/TCF transcriptional activity. Results are normalized to saline controls. Mean ± SEM of 3 experiments. (B) Quantitation of Sox9+ neural crest cells following the indicated treatments. Pretreatment with Bapta-AM normalized the number of Sox9+ neural crest in ethanol-treated hindbrain. Mean ± SEM of 3–4 embryos per group. (C) Quantitation of LTR+Sox9+ neural crest following the indicated treatments. Pretreatment with Bapta-AM significantly reduced the number of apoptotic neural crest in ethanol-treated hindbrain to normal levels. Mean ± SEM of 3–4 embryos per group. (A, B) were analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s post hoc analysis. (C) was analyzed using one-way ANOVA and Student-Newman-Kuels post-hoc analysis. *p<0.005 vs. saline, + p<0.05 vs. ethanol, ++ p<0.01 vs. ethanol.
Figure 9
Figure 9
Ethanol signaling pathway that initiates neural crest apoptosis. Shown is a summary of published studies (Debelak-Kragtorp et al. 2003; Garic-Stankovic et al. 2005, 2006) and results herein. Ethanol interacts with a G-protein-coupled receptor (GPCR) of unknown identity to activate Gαi2/3 and Gβγ. Within seconds, the latter stimulates Phospholipase Cβ-mediated synthesis of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium release predominantly from intracellular stores. The mobilized calcium interacts with one of several possible effectors, including CaMKII, Protein Kinase C or calpain to destabilize β-catenin and terminate its transcriptional stimulation of the TCF/LEF complex. Alternately, ethanol may destabilize β-catenin through its potential stimulation of GSK3b. The loss of β-catenin’s transcriptional activity promotes apoptosis whereas its maintenance promotes the survival of neural crest and neuronal precursors. “?” indicates that the contribution of this signal to ethanol-mediated cell death is unknown at this time.

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