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The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Parsons MJ, Brancaccio M, Sethi S, Maywood ES, Satija R, Edwards JK, Jagannath A, Couch Y, Finelli MJ, Smyllie NJ, Esapa C, Butler R, Barnard AR, Chesham JE, Saito S, Joynson G, Wells S, Foster RG, Oliver PL, Simon MM, Mallon AM, Hastings MH, Nolan PM - Cell (2015)

Bottom Line: Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed.Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices.In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

View Article: PubMed Central - PubMed

Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.

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Significant Decreases in Neuropeptide Expression Detected in Zfhx3Sci/+ SCN(A and B) mRNA expression of both (A) Vip and (B) Grp was significantly decreased in SCN of Zfhx3Sci/+ (gray lines) compared to Zfhx3+/+ (black lines) at multiple time points (n = 4). p < 0.05, ANOVA.(C) Zfhx3 mRNA expression in SCN is stable throughout the day and does not significantly differ by genotype (n = 4, gray and black lines represent Zfhx3Sci/+ and Zfhx3+/+ respectively).(D) ZFHX3 protein localization does not grossly differ across genotype at ZT6.(E and F) As shown here, (E) VIP and (F) GRP immunofluorescence in SCN was decreased in Zfhx3Sci/+ animals (p < 0.05).(G and H) As shown here, (G) VIPR2 and (H) AVP SCN protein expression was not different between genotypes (p > 0.1).(I) Confocal images (20× magnification) of immunostaining for VIP, GRP, and DAPI and the composite image in the SCN of Zfhx3+/+ (top panel) and Zfhx3Sci/+ (bottom panel). Scale bars, 100 μm.(J and K) The number of (J) VIP- and (K) AVP-immunopositive cells was quantified, with no significant differences detected across genotype.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S3.
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fig4: Significant Decreases in Neuropeptide Expression Detected in Zfhx3Sci/+ SCN(A and B) mRNA expression of both (A) Vip and (B) Grp was significantly decreased in SCN of Zfhx3Sci/+ (gray lines) compared to Zfhx3+/+ (black lines) at multiple time points (n = 4). p < 0.05, ANOVA.(C) Zfhx3 mRNA expression in SCN is stable throughout the day and does not significantly differ by genotype (n = 4, gray and black lines represent Zfhx3Sci/+ and Zfhx3+/+ respectively).(D) ZFHX3 protein localization does not grossly differ across genotype at ZT6.(E and F) As shown here, (E) VIP and (F) GRP immunofluorescence in SCN was decreased in Zfhx3Sci/+ animals (p < 0.05).(G and H) As shown here, (G) VIPR2 and (H) AVP SCN protein expression was not different between genotypes (p > 0.1).(I) Confocal images (20× magnification) of immunostaining for VIP, GRP, and DAPI and the composite image in the SCN of Zfhx3+/+ (top panel) and Zfhx3Sci/+ (bottom panel). Scale bars, 100 μm.(J and K) The number of (J) VIP- and (K) AVP-immunopositive cells was quantified, with no significant differences detected across genotype.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S3.

Mentions: A comparison of module 1 elements in Zfhx3Sci/+ and Zfhx3+/+ SCN revealed that 15 of 21 genes showed decreased expression in mutants. This included a number of neuropeptides and their receptors, such as: arginine vasopressin (Avp), gastrin-releasing peptide (Grp), Nms, prokineticin 2 (Prok2), Prokr2, Vip, and Vipr2. We measured their SCN mRNA expression at six time points across the light:dark cycle, using qPCR. Grp, Vip, and Vipr2 had damped mRNA expression in Zfhx3Sci/+ SCN (two-way ANOVA, genotype main effect, p < 0.05). Expression of Grp and Vip, two neuropeptides previously shown to be important for regulating the firing patterns of SCN neurons (Aton et al., 2005; Brown et al., 2005; Maywood et al., 2006;), was decreased but not absent at most time points (Figures 4A and 4B). It should be noted that Zfhx3 expression is not cyclic in the SCN, with no significant differences in mRNA expression (two-way ANOVA, time main effect, p > 0.2) and no gross differences in protein localization within the SCN between Zfhx3Sci/+ and Zfhx3+/+ animals (Figures 4C and 4D).


The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Parsons MJ, Brancaccio M, Sethi S, Maywood ES, Satija R, Edwards JK, Jagannath A, Couch Y, Finelli MJ, Smyllie NJ, Esapa C, Butler R, Barnard AR, Chesham JE, Saito S, Joynson G, Wells S, Foster RG, Oliver PL, Simon MM, Mallon AM, Hastings MH, Nolan PM - Cell (2015)

Significant Decreases in Neuropeptide Expression Detected in Zfhx3Sci/+ SCN(A and B) mRNA expression of both (A) Vip and (B) Grp was significantly decreased in SCN of Zfhx3Sci/+ (gray lines) compared to Zfhx3+/+ (black lines) at multiple time points (n = 4). p < 0.05, ANOVA.(C) Zfhx3 mRNA expression in SCN is stable throughout the day and does not significantly differ by genotype (n = 4, gray and black lines represent Zfhx3Sci/+ and Zfhx3+/+ respectively).(D) ZFHX3 protein localization does not grossly differ across genotype at ZT6.(E and F) As shown here, (E) VIP and (F) GRP immunofluorescence in SCN was decreased in Zfhx3Sci/+ animals (p < 0.05).(G and H) As shown here, (G) VIPR2 and (H) AVP SCN protein expression was not different between genotypes (p > 0.1).(I) Confocal images (20× magnification) of immunostaining for VIP, GRP, and DAPI and the composite image in the SCN of Zfhx3+/+ (top panel) and Zfhx3Sci/+ (bottom panel). Scale bars, 100 μm.(J and K) The number of (J) VIP- and (K) AVP-immunopositive cells was quantified, with no significant differences detected across genotype.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S3.
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fig4: Significant Decreases in Neuropeptide Expression Detected in Zfhx3Sci/+ SCN(A and B) mRNA expression of both (A) Vip and (B) Grp was significantly decreased in SCN of Zfhx3Sci/+ (gray lines) compared to Zfhx3+/+ (black lines) at multiple time points (n = 4). p < 0.05, ANOVA.(C) Zfhx3 mRNA expression in SCN is stable throughout the day and does not significantly differ by genotype (n = 4, gray and black lines represent Zfhx3Sci/+ and Zfhx3+/+ respectively).(D) ZFHX3 protein localization does not grossly differ across genotype at ZT6.(E and F) As shown here, (E) VIP and (F) GRP immunofluorescence in SCN was decreased in Zfhx3Sci/+ animals (p < 0.05).(G and H) As shown here, (G) VIPR2 and (H) AVP SCN protein expression was not different between genotypes (p > 0.1).(I) Confocal images (20× magnification) of immunostaining for VIP, GRP, and DAPI and the composite image in the SCN of Zfhx3+/+ (top panel) and Zfhx3Sci/+ (bottom panel). Scale bars, 100 μm.(J and K) The number of (J) VIP- and (K) AVP-immunopositive cells was quantified, with no significant differences detected across genotype.Error bars indicate SEM. ∗p < 0.05, t test.See also Figure S3.
Mentions: A comparison of module 1 elements in Zfhx3Sci/+ and Zfhx3+/+ SCN revealed that 15 of 21 genes showed decreased expression in mutants. This included a number of neuropeptides and their receptors, such as: arginine vasopressin (Avp), gastrin-releasing peptide (Grp), Nms, prokineticin 2 (Prok2), Prokr2, Vip, and Vipr2. We measured their SCN mRNA expression at six time points across the light:dark cycle, using qPCR. Grp, Vip, and Vipr2 had damped mRNA expression in Zfhx3Sci/+ SCN (two-way ANOVA, genotype main effect, p < 0.05). Expression of Grp and Vip, two neuropeptides previously shown to be important for regulating the firing patterns of SCN neurons (Aton et al., 2005; Brown et al., 2005; Maywood et al., 2006;), was decreased but not absent at most time points (Figures 4A and 4B). It should be noted that Zfhx3 expression is not cyclic in the SCN, with no significant differences in mRNA expression (two-way ANOVA, time main effect, p > 0.2) and no gross differences in protein localization within the SCN between Zfhx3Sci/+ and Zfhx3+/+ animals (Figures 4C and 4D).

Bottom Line: Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed.Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices.In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

View Article: PubMed Central - PubMed

Affiliation: MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK.

Show MeSH