1. Volume 20, Issue 9, Pages 829-835 (May 2010)
Identification of Eya3 and TAC1 as Long-Day Signals in the Sheep Pituitary 
Sandrine M. Dupré, Katarzyna Miedzinska, Chloe V. Duval, Le Yu, Robert L. Goodman, Gerald A. Lincoln, Julian R.E. Davis, Alan S. McNeilly, David D. Burt, Andrew S.I. Loudon 
Current Biology 
Volume 20, Issue 9, Pages (May 2010)
DOI: /j.cub
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2. Figure 1
Transfer from Short Photoperiod to 7 or 28 Days in Long Photoperiod Significantly Alters Circulating Prolactin Blood Levels and Gene Expression in the Pars Tuberalis in Sheep
(A) Prolactin (PRL) concentrations in short photoperiod (SP) and after 7 and 28 days in long photoperiod (7LP and 28LP), shown as mean ± standard error of the mean (SEM). Thirty sheep were maintained under artificial SP. After 8 weeks, five sheep were killed either 4 hr after light onset or 4 hr after dark onset (arrowheads). The rest of the cohort (n = 20) was transferred to LP, and groups of five animals were killed either 7 or 28 days after the transfer (again, 4 hr after light onset and 4 hr after dark onset [arrowheads]).
(B) Volcano plots representing the alteration in gene expression in the pars tuberalis (PT) after 7 and 28 days in LP, with the ratio of expression in SP over LP on the x axis and the significance of the changes with a false discovery rate (FDR) threshold (red horizontal line) set at 10% on the y axis. Eya3 and TAC1, indicated by the arrows, show significant alteration in expression after 7 and 28 days in LP for Eya3 and after 28 days in LP for TAC1. See Table S2 for lists of significantly altered genes in LP.
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3. Figure 2
Quantification of Eya3 mRNA Expression in the Sheep Pars Tuberalis
Quantification and representative expression of Eya3 in the sheep PT in 20 μm coronal cryostat sections. Values are shown as mean ± SEM. Statistical significance was assessed by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test for time of day and photoperiodic effect with ∗∗∗p < (A) and one-way ANOVA followed by Bonferroni post hoc test for ZT3 (∗∗∗p < 0.001) and ZT15 (###p < 0.001) compared to the other time points in SP (B) and LP (C). Scale bars represent 200 μm.
(A) Eya3 shows significant upregulation in the sheep PT in the light phase (ZT4) on day 1 and day 7 of LP exposure.
(B) Eya3 shows low levels of expression in the PT under SP conditions with no significant variation over 24 hr. The inset shows expression at ZT3.
(C) On day 28 of LP, sampling over a 24 hr period shows that Eya3 expression is biphasic with peaks at the early and late light phase (ZT3 and ZT15). The inset shows expression at ZT3.
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4. Figure 3
Quantification of TAC1 mRNA Expression in the Sheep Pars Tuberalis
Quantification and representative expression of TAC1 in the sheep PT in 20 μm coronal cryostat sections. Values are shown as mean ± SEM. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post hoc test for time of day and photoperiodic effect with ∗∗∗p < (A) and one-way ANOVA followed by Bonferroni post hoc test for ZT3 (∗p < 0.05, ∗∗∗p < 0.001) compared to the other time points in SP (B) and LP (C). Scale bars represent 200 μm.
(A) TAC1 expression is upregulated in the light phase (ZT4) in LP compared to SP with a progressive rise from day 1 in LP to day 7 in LP.
(B) TAC1 expression could not be detected in the PT under SP conditions. The dashed horizontal line represents nonspecific background expression as observed with a sense probe. The inset shows expression at ZT3.
(C) TAC1 is strongly expressed in the light phase on day 28 of LP exposure with a peak in the early light phase and a progressive decline toward the end of the day. The inset shows expression at ZT3.
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5. Figure 4
Substance P and Neurokinin A Are Present in the Ovine Pars Tuberalis and Act as Potential Secretagogues on Pituitary Cells
(A–H) Immunohistochemistry for substance P (A–D) and neurokinin A (NKA) (E–H) showing that both peptides are expressed in the PT. Preadsorption of the antibodies with blocking peptide for substance P (B) or NKA (F) shows no specific staining. The dashed line in (A) represents the limit between the PT and the ME (median eminence). Scale bars represent 500 μm in (A), (B), (E), and (F), 125 μm in (C) and (G), and 72.5 μm in (D) and (H).
(I) Radioimmunoassays for PRL secretion from dispersed ovine pituitary cells after 1 hr treatment with 10 μM forskolin (FSK), NKA, substance P (SP), and a short fragment of substance P (SP1-7) at 10−9 M. Values are shown as mean ± SEM. Statistical significance against untreated control (Ctl) was set at ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < by one-way ANOVA followed by Bonferroni post hoc test.
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6. Figure 5
Characterization of the Cell Types Expressing Neurokinin Receptors in the Ovine Pars Distalis
Double immunofluorescence showing expression of NK1, 2, and 3R (red) in the main pituitary cell types (green): lactotrophs (PRL), gonadotrophs (LH), corticotrophs (ACTH), somatotrophs (GH), thyrotrophs (TSH), and folliculostellate cells (S100). NK1R is colocalized with ACTH and S100; NK2R with LH, ACTH, and TSH; and NK3R with ACTH as shown in the insets. Scale bars represent 20 μm in the main panels and 10 μm in the insets. See Table S3 for percentages of pituitary cell types coexpressing NK1–3R.
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