Identification of Eya3 and TAC1 As Long-Day Signals in the Sheep

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Current Biology 20, 829–835, May 11, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.02.066 Report Identiﬁcation of Eya3 and TAC1 as Long-Day Signals in the Sheep Pituitary

Sandrine M. Dupre´,1 Katarzyna Miedzinska,2 Chloe V. Duval,1 photoperiod change to local hypothalamic pathways govern- Le Yu,3 Robert L. Goodman,4 Gerald A. Lincoln,5 ing reproductive changes [3]. Recent studies in the Japanese Julian R.E. Davis,6 Alan S. McNeilly,2 David D. Burt,3,* quail examined the pattern of gene activation following expo- and Andrew S.I. Loudon1,* sure to reproductive stimulatory long photoperiod (LP). These 1Faculty of Life Sciences, University of Manchester, studies revealed that thyrotropin beta subunit (TSHb) and Manchester M13 9PT, UK the transcriptional cofactor eyes absent 3 (Eya3) are the first 2Medical Research Council Human Reproductive Sciences genes activated in the PT on day 1 following LP exposure [8]. Unit, Centre for Reproductive Biology, The Queen’s Medical In seasonal mammals, the PT is also known to play a direct Research Institute, 47 Little France Crescent, Edinburgh EH16 role in the regulation of annual prolactin (PRL) cycle. This 4TJ, UK hormone is strongly photoperiodic, with secretion rising over 3The Roslin Institute, University of Edinburgh, the first few days of LP exposure [9]. Studies in both sheep Roslin BioCentre, Roslin, Midlothian EH25 9PS, UK and seasonal rodent models (Syrian hamsters) show that 4Department of Physiology and Pharmacology, West Virginia ex vivo PT cultures secrete one or more low-molecular-weight University School of Medicine, One Medical Center Drive, (1–10 kDa) peptidergic PRL-releasing factors (termed ‘‘tubera- PO Box 9229, Health Sciences Center, Morgantown, lins’’), which drive seasonal PRL secretion from dispersed WV 26505-9229, USA cultures of anterior pituitary cells [5–7], and tuberalin activity 5Reproductive and Developmental Sciences, Centre for is modified by the prior photoperiodic regime that animals Reproductive Biology, University of Edinburgh, Edinburgh are exposed to (augmented during LP exposure [7]). Further- EH16 4TJ, UK more, hypothalamic disconnection experiments in sheep 6Endocrine Sciences Research Group, Manchester Academic demonstrate robust photoperiodic rhythms of PRL following Health Science Centre, University of Manchester, Manchester severance of the anatomical links between the pituitary and M13 9PT, UK secretory nerve terminals of the hypothalamus [10]. Thus, seasonal PRL response depends on a local paracrine PRL- stimulating factor of PT origin acting on distal lactotroph Summary cells. This second circuit has thus far been described only in mammals, and the identity (or identities) of tuberalin has Seasonally breeding mammals such as sheep use photope- remained enigmatic. riod, encoded by the nocturnal secretion of the pineal hormone melatonin, as a critical cue to drive hormone Array Studies Reveal a Novel PT-Activated Gene in Sheep rhythms and synchronize reproduction to the most optimal following LP Stimulation time of year [1, 2]. Melatonin acts directly on the pars tubera- To identify circuits and possible PRL-regulating genes lis (PT) of the pituitary, regulating expression of thyrotropin, activated in the PT following LP stimulation, we collected which then relays messages back to the hypothalamus to sheep PT tissue 4 hr after lights on (ZT4) or off (ZT20) after 7 control reproductive circuits [3, 4]. In addition, a second or 28 days of LP exposure and compared transcript levels local intrapituitary circuit controls seasonal prolactin (PRL) with those of PT from short photoperiod (SP)-housed sheep release via one or more currently uncharacterized low- culled at the same time points (ZT4 and ZT12) (Figure 1A). molecular-weight peptides, termed ‘‘tuberalins,’’ of PT origin These times of collection were selected to match to the earliest [5–7]. Studies in birds have identified the transcription factor time when robust PRL responses are detected (day 7) and Eya3 as the first molecular response activated by long photo- when PRL responses are close to maximal (day 28) [4, 9, 10], period (LP) [8]. Using arrays and in situ hybridization studies, as shown in Figure 1A. Using a bovine cDNA array previously we demonstrate here that Eya3 is the strongest LP-activated validated for use in sheep [11], we screened for altered gene in sheep, revealing a common photoperiodic molecular expression of transcripts at each of the above time points response in birds and mammals. We also demonstrate TAC1 following LP exposure and compared them against SP values. (encoding the tachykinins substance P and neurokinin A) to The comparison involved hybridization against a common be strongly activated by LP within the sheep PT. We show reference pool (see Supplemental Experimental Procedures that these PRL secretagogues act on primary pituitary cells available online) and as a result was likely to detect only and thus are candidates for the elusive PT-expressed tuber- strongly differentially expressed genes. Bioinformatic anal- alin seasonal hormone regulator. yses revealed that the majority of significant changes in gene expression were observed during the light phase of LP (ZT4) Results and Discussion (Table S1). We therefore focused our interest on changes occurring at day 7 and day 28 at ZT4 to annotate significantly Seasonal Neuroendocrine Regulation by the Pars altered genes and found 98 known genes altered in expression Tuberalis at day 7 (64 increased, 34 suppressed) and 18 genes at day 28 In birds and mammals, the pars tuberalis (PT) operates (16 increased and 2 suppressed) (Figure 1B; Table S2). We as a crucial relay conveying thyroid-dependent effects of compared our data on transcripts exhibiting altered expression in the PT with data sets of genes known to exhibit *Correspondence: [email protected] (D.D.B.), andrew.loudon@ strong circadian expression in several different tissues in the manchester.ac.uk (A.S.I.L.) mouse (suprachiasmatic nucleus, kidney, liver, and aorta) Current Biology Vol 20 No 9 830

statistical power to detect cycling revealed that approximately 7% of transcripts cycled in liver and 1% in pituitary [14]. Our data thus reveal a remarkable enrichment of circadian transcripts in the PT and highlight the likely critical importance of the circadian clockwork in encoding seasonal time [15].

Long-Day Activation of Eya3 in the Ovine PT On day 7 and day 28, the most strongly upregulated gene in the array was Eya3 (Figure 1B). In order to define tissue expression with higher-resolution quantification and to determine the time course of Eya3 upregulation in the sheep PT, we examined its expression by in situ hybridization in sheep. For this purpose, we set up another cohort of animals kept in SP and then exposed to 1 or 7 days of LP stimulation and culled 4 hr into the light phase (ZT4) or 4 hr into the dark phase (ZT12 or 20 for SP- or LP-housed animals, respectively; Figure 2A). Eya3 expression was specific to the PT region, with no detectable hybridization signal in the adjacent hypothalamus (Figures 2A–2C) or distal pituitary (data not shown). In SP, a hybridization signal was detected, with no change between ZT4 and ZT12. Upon transferring the animals to LP, we observed an abrupt increase in expression at ZT4 on day 1, which was sustained at day 7 (Figure 2A). In order to define more precisely the temporal expression of Eya3, we collected samples over a 24 hr period at 4 hr intervals from SP-housed sheep, or 28 days after LP exposure. In SP, Eya3 expression showed no significant variation over 24 hr (Figure 2B). In contrast, in LP, we observed a strong early-day peak (as seen above on days 1 and 7) as well as a second peak in the late light phase (Figure 2C). These results confirmed the array results on days 7 and 28 but also provided information indicating that Eya3 acts as an acute response to photoperiod with an upregulation on day 1 onward (Figure 2A). These data parallel an earlier study in quail in which Eya3 was identified as one of the first long-day response genes in the avian PT [8]. Compar- ison of LP-activated genes here with the quail data revealed that Eya3 was the only gene common to both data sets. In our study, Eya3 was the most strongly differentially expressed transcript on the entire array (over 3- to 4-fold; Figure 1B; Table S2). We show here that Eya3 is activated during the first Figure 1. Transfer from Short Photoperiod to 7 or 28 Days in Long Photope- days of exposure to LP, as found in the quail model; however, riod Significantly Alters Circulating Prolactin Blood Levels and Gene in sheep, Eya3 continues to provide a sustained marker for LP Expression in the Pars Tuberalis in Sheep activation many weeks later. Interestingly, we also identified (A) Prolactin (PRL) concentrations in short photoperiod (SP) and after 7 and a strong biphasic pattern of expression in LP with a peak in 28 days in long photoperiod (7LP and 28LP), shown as mean 6 standard error of the mean (SEM). Thirty sheep were maintained under artificial SP. the early and late light phase (Figures 2A and 2C). 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 Identification of a Prolactin-Regulating Pathway LP, and groups of five animals were killed either 7 or 28 days after the trans- Since the identification of factors released by the PT driving fer (again, 4 hr after light onset and 4 hr after dark onset [arrowheads]). the seasonal PRL pituitary secretion in seasonal mammals, (B) Volcano plots representing the alteration in gene expression in the pars the existence of tuberalins has remained elusive. We next 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 screened our array data for differentially expressed genes discovery rate (FDR) threshold (red horizontal line) set at 10% on the coding for putative PRL secretagogues. We identified TAC1 y axis. Eya3 and TAC1, indicated by the arrows, show significant alteration (tachykinin precursor 1; Figure 1B; Table S2) as the only poten- in expression after 7 and 28 days in LP for Eya3 and after 28 days in LP for tial candidate gene showing significant upregulation on day TAC1. See Table S2 for lists of significantly altered genes in LP. 28. TAC1 encodes tachykinins (reviewed in [16]), bioactive peptides including substance P and neurokinin A (NKA), which have been shown to be capable of regulating PRL release [12, 13]. This subset of mouse cycling genes thus represents in vivo (reviewed in [17]). Moreover, these peptides are in the a core group of clock-controlled genes likely to cycle in most correct molecular weight range (1–10 kDa) to act as candidate body tissues. A comparison with ovine PT transcripts revealed tuberalins [6]. that approximately 50% of these genes with altered expres- We used in situ hybridization for ovine TAC1 in the PT sion in LP were also cyclic in mouse (Table S2). In mice, the to determine more precisely the onset of the effect of LP expo- overall proportions of cycling transcripts in different body sure on its expression. TAC1 has been previously reported to organs varies, but an extensive recent study using appropriate be expressed in the hypothalamus at the mRNA level (in rat, Encoding Long-Day Signals in the Sheep Pituitary 831

Figure 2. Quantification of Eya3 mRNA Expression in the Sheep Pars Tuberalis Quantification and representative expression of Eya3 in the sheep PT in 20 mm coronal cryostat sections. Values are shown as mean 6 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 < 0.001 (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 mm. (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. human, and monkey [18, 19]), and we confirmed this in sheep pituitary tissue and treated dispersed cells with NKA or (Figures 3A–3C) with no significant variation across time of day substance P, using the adenylate cyclase activator forskolin or photoperiod. In the PT, we did not observe a detectable as a positive control (Figure 4I). Because substance P is known hybridization signal under SP conditions (Figure 3A). However, to be cleaved by endopeptidases, we tested here a known following exposure to LP, we observed induction during the bioactive cleavage product of substance P, substance P 1-7 light phase that was moderate on day 1 and significantly (SP1-7) [22](Figure 4I). NKA treatment triggered a strong stronger (4- to 5-fold induction) on day 7 in the PT (Figure 3A). induction of PRL secretion that was of similar magnitude to We next investigated the temporal expression of TAC1 in the that observed following forskolin treatment. Although our PT by using sheep kept in SP or in LP for 28 days as described data showed no significant effect of substance P on PRL above. Here, TAC1 expression exhibited a 10-fold induction at release, SP1-7 caused a significant stimulatory effect (Fig- the onset of the day, with gradual decrease of expression over ure 4I). The action of SP1-7 compared to SP itself suggests the light phase (Figure 3C). These data reveal that TAC1 is the importance of substance P preprocessing by endopepti- induced in the ovine PT by LP exposure with a gradually dases within the PT. We have recently undertaken a screen augmented response from day 1 to day 7 (Figure 3A) and for mRNA expressed in the PT via ‘‘deep sequencing’’ Illumina day 28 (Figure 3C), matching the rise in PRL secretion over next-generation sequencing technology and have identified the same time course. endopeptidase 24.11, responsible for the cleavage of substance P into SP1-7 [22], as being expressed in this tissue Pituitary Responses to Tachykinins (D.D.B., L.Y., S.M.D., A.S.M., and A.S.I.L., unpublished data). Earlier studies reported substance P and NKA in the ovine PT This implies that substance P could be activated in the PT and suggested that these peptides could act as PRL secreta- itself. Tachykinins have been described in the hypothalamus gogues [20, 21]. Previous studies in rodents showed that and the PD in mammals, and their role in PRL regulation has substance P and NKA can act as potent regulators of pituitary been the focus of several studies (reviewed in [17]). Our data hormone secretion. Using sheep as a model, we first con- now provide evidence for specific photoperiod-dependent firmed the localization of both peptides in the PT by immuno- expression of TAC1 in the PT, consistent with the hypothesis histochemistry (Figures 4A–4H) and then investigated their that products of the TAC1 gene (substance P and NKA) may effects on the pars distalis (PD) cells. We collected ovine act as a seasonal PRL regulator in distal lactotroph cells. Current Biology Vol 20 No 9 832

Figure 3. Quantification of TAC1 mRNA Expression in the Sheep Pars Tuberalis Quantification and representative expression of TAC1 in the sheep PT in 20 mm coronal cryostat sections. Values are shown as mean 6 SEM. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post hoc test for time of day and photoperiodic effect with ***p < 0.001 (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 mm. (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.

Expression of Neurokinin Receptors control may involve an additional intrapituitary signaling path- Three cognate G protein receptors have been described for way, with NKA and activated substance P acting on other secre- tachykinins (NK1–3R, binding with differing afﬁnities to sub- tory cell types, which may release paracrine factors controlling stance P and NKA in the order NK1R, substance P > NKA; hormone secretion from neighboring lactotrophs (Figure S1). NK2R, NKA > substance P; NK3R, NKA > substance P [23]). Several studies in sheep have shown that prolactin is secreted Using immunohistochemistry, we examined expression pat- in a diurnal pattern, with elevated levels in the late day/nocturnal terns for these receptors in the sheep PD (Figure 5). This period [25]. Our data here imply that the prolactin-releasing revealed that NK1–3R were all expressed in the hormone- action of tachykinins is likely mediated via intermediate cell secreting cells of the sheep PD (Figure 5). However, colocaliza- types with unknown temporal dynamics in terms of de novo tion patterns for each of the main pituitary secretory cell types RNA transcription and/or local hormone release in target cells (lactotroph, somatotroph, gonadotroph, thyrotroph, and corti- of the PD. Thus, rhythmic TAC1 transcripts in the PT over the cotroph) and folliculostellate cells revealed that the major light phase may be phase-lagged relative to circulating patterns receptor for NKA (NK2R) was localized clearly in corticotrophs of prolactin. Several NKR-positive cell types are attractive and gonadotrophs and at a low level in thyrotrophs and that candidates as intermediate prolactin-regulating cells. For the major receptor for substance P (NK1R) was localized in instance, corticotroph cells strongly coexpress all three NKRs corticotrophs and folliculostellate cells. Although expression and secrete a complex range of pro-opiomelanocortin-derived of NK2R has previously been reported in a subset of lactotroph gene products, some capable of driving PRL secretion. Further- cells in rat PD cultures [24], we were unable to identify coloc- more, (NK1R-positive) folliculostellate cells are widely distrib- alization of NK1–3R in lactotrophs, indicating that the effect uted throughout the PD and also the PT and have been impli- of these peptides on PRL secretion is likely to be indirect. cated in serving a role as both local pituitary stem cells and Our results also indicate colocalization for NK1R with cortico- paracrine regulators [26]. Our data are thus compatible with troph and folliculostellate cells and for NK3R with gonado- a model in which seasonal PRL regulation represents a distinc- troph cells (Table S3). tive pituitary circuit separate from that controlling thyrotropin Lack of colocalization of tachykinin receptors (NK1–3R) to and reproductive changes (Figure S1). Whether such a circuit cells of the lactotroph cell lineage suggests that seasonal PRL operates in birds has yet to be determined. Encoding Long-Day Signals in the Sheep Pituitary 833

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 Figure 5. Characterization of the Cell Types Expressing Neurokinin Recep- the PT and the ME (median eminence). Scale bars represent 500 mm in (A), tors in the Ovine Pars Distalis (B), (E), and (F), 125 mm in (C) and (G), and 72.5 mm in (D) and (H). (I) Radioimmunoassays for PRL secretion from dispersed ovine pituitary Double immunofluorescence showing expression of NK1, 2, and 3R (red) in cells after 1 hr treatment with 10 mM forskolin (FSK), NKA, substance P the main pituitary cell types (green): lactotrophs (PRL), gonadotrophs (LH), (SP), and a short fragment of substance P (SP1-7) at 1029 M. Values are corticotrophs (ACTH), somatotrophs (GH), thyrotrophs (TSH), and folliculos- shown as mean 6 SEM. Statistical significance against untreated control tellate cells (S100). NK1R is colocalized with ACTH and S100; NK2R with LH, (Ctl) was set at *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA fol- ACTH, and TSH; and NK3R with ACTH as shown in the insets. Scale bars lowed by Bonferroni post hoc test. represent 20 mm in the main panels and 10 mm in the insets. See Table S3 for percentages of pituitary cell types coexpressing NK1–3R.

Dual Role of PT Thyrotrophs in Controlling Seasonal as part of an evolutionarily conserved gene network in Hormone Release Drosophila and vertebrates, controlling tissue development The present study identiﬁes Eya3 as the ﬁrst common including in the eye. EYA3 has recently been shown to act as long-day molecular signature for birds and mammals in the a key regulator of DNA breakpoint repair [28]; it is plausible PT. EYA operates as both a transcriptional cofactor and an to propose that long-term seasonal changes in neuroendo- enzyme (tyrosine phosphatase) [27]. The regulatory potential crine control may depend in part on local tissue photope- of such a bifunctional protein is considerable; Eya3 has an riod-driven remodeling events in the PT (as demonstrated in important role in photoreceptor differentiation and operates the ependymal region of quail [29]), which within the PT may Current Biology Vol 20 No 9 834

be controlled by an EYA3-driven pathway. The extent to which 4. Hanon, E.A., Lincoln, G.A., Fustin, J.M., Dardente, H., Masson-Pe´ vet, EYA3 might be directly involved in the regulation of TAC1 and M., Morgan, P.J., and Hazlerigg, D.G. (2008). Ancestral TSH mechanism TSHb gene expression has yet to be determined (Figure S1). signals summer in a photoperiodic mammal. Curr. Biol. 18, 1147–1152. 5. Graham, E.S., Webster, C.A., Hazlerigg, D.G., and Morgan, P.J. (2002). PRL has a unique seasonal signature in all vertebrate Evidence for the biosynthesis of a prolactin-releasing factor from the species in that it is consistently elevated by LP stimulation ovine pars tuberalis, which is distinct from thyrotropin-releasing [30]. As such, it operates as the only true LP-regulated hormone. J. Neuroendocrinol. 14, 945–954. hormone. In contrast, pituitary hormones regulating reproduc- 6. Morgan, P.J., Webster, C.A., Mercer, J.G., Ross, A.W., Hazlerigg, D.G., tive responses are tuned to the breeding biology of the species MacLean, A., and Barrett, P. (1996b). The ovine pars tuberalis secretes and may be activated in spring (rodents) or autumn (sheep). a factor(s) that regulates gene expression in both lactotropic and non- lactotropic pituitary cells. Endocrinology 137, 4018–4026. PRL is known to control the molt cycle of hair and feather 7. Stirland, J.A., Johnston, J.D., Cagampang, F.R.A., Morgan, P.J., Castro, follicle growth in mammals and birds, and these are strictly M.G., White, M.R.H., Davis, J.R.E., and Loudon, A.S.I. (2001). Photope- seasonal events with similar timing in most habitats irrespec- riodic regulation of prolactin gene expression in the Syrian hamster by tive of breeding season (Figure S1). Accordingly, the system a pars tuberalis-derived factor. J. Neuroendocrinol. 13, 147–157. we have defined here may operate as a form of seasonal 8. Nakao, N., Ono, H., Yamamura, T., Anraku, T., Takagi, T., Higashi, K., hourglass, driving annual cycles of PRL to track predictable Yasuo, S., Katou, Y., Kageyama, S., Uno, Y., et al. (2008). Thyrotrophin photoperiod changes. Earlier studies predicted that a photo- in the pars tuberalis triggers photoperiodic response. Nature 452, 317–322. period-regulated PRL-secreting pathway should be character- 9. Hazlerigg, D.G., Andersson, H., Johnston, J.D., and Lincoln, G. (2004). ized by the identification of a low-molecular-weight peptide Molecular characterization of the long-day response in the Soay sheep, within the PT region of the pituitary [6] and that the gene en- a seasonal mammal. Curr. Biol. 14, 334–339. coding this factor should be activated in a PT-specific manner 10. Lincoln, G.A., and Clarke, I.J. (1994). Photoperiodically-induced cycles by photoperiodic stimulation, in a pattern consistent with the in the secretion of prolactin in hypothalamo-pituitary disconnected natural rise in PRL secretion detected in peripheral blood in rams: Evidence for translation of the melatonin signal in the pituitary gland. J. Neuroendocrinol. 6, 251–260. the weeks following stimulation by LP. A further prediction is 11. Dupre´ , S.M., Burt, D.W., Talbot, R., Downing, A., Mouzaki, D., Wadding- that this candidate peptide should be capable of eliciting ton, D., Malpaux, B., Davis, J.R., Lincoln, G.A., and Loudon, A.S. (2008). PRL release from primary pituitary cells in the same species. Identification of melatonin-regulated genes in the ovine pituitary pars The data we present are therefore consistent with the hypoth- tuberalis, a target site for seasonal hormone control. Endocrinology esis that tachykinins may serve as the elusive seasonal tuber- 149, 5527–5539. alins driving PRL secretion. Testing the biological action of 12. Panda, S., Antoch, M.P., Miller, B.H., Su, A.I., Schook, A.B., Straume, M., Schultz, P.G., Kay, S.A., Takahashi, J.S., and Hogenesch, J.B. (2002). tachykinins in vivo is likely to be highly technically demanding, Coordinated transcription of key pathways in the mouse by the circa- given the short half-life of these peptides in circulation and the dian clock. Cell 109, 307–320. need for local enzymatic processing, and may require direct 13. Rudic, R.D., McNamara, P., Reilly, D., Grosser, T., Curtis, A.M., Price, in vivo targeting of pituitary structures. T.S., Panda, S., Hogenesch, J.B., and FitzGerald, G.A. (2005). Bioinfor- matic analysis of circadian gene oscillation in mouse aorta. Circulation 112, 2716–2724. Supplemental Information 14. Hughes, M.E., DiTacchio, L., Hayes, K.R., Vollmers, C., Pulivarthy, S., Baggs, J.E., Panda, S., and Hogenesch, J.B. (2009). Harmonics of circa- Supplemental Information includes three tables, one figure, and Supple- dian gene transcription in mammals. PLoS Genet. 5, e1000442. mental Experimental Procedures and can be found with this article online 15. Lincoln, G.A., Andersson, H., and Loudon, A. (2003). Clock genes in at doi:10.1016/j.cub.2010.02.066. calendar cells as the basis of annual timekeeping in mammals— a unifying hypothesis. J. Endocrinol. 179, 1–13. Acknowledgments 16. Page, N.M. (2005). New challenges in the study of the mammalian tachykinins. Peptides 26, 1356–1368. We thank J. Docherty and staff at the Marshall Building (University of Edin- 17. Debeljuk, L., and Lasaga, M. (2006). Tachykinins and the control of burgh) for animal care; R. Talbot and A. Downing at the ARK-Genomics prolactin secretion. Peptides 27, 3007–3019. facility (Roslin Institute) for support in conducting the microarray experi- 18. Gautreau, A., Duval, P., and Kerdelhue´ , B. (1997). Variations in levels of ment; A.C. West (University of Manchester) for assistance with tissue collec- substance P-encoding beta-, gamma-preprotachykinin and substance tion; and B.R.C. Saer (University of Manchester) for assistance with tissue P receptor NK-1 transcripts in the rat hypothalamus throughout the dissection and in situ hybridization experiments. This work was supported estrous cycle: A correlation between amounts of beta-preprotachykinin by Biotechnology and Biological Sciences Research Council (UK) grant and NK-1 mRNA. Brain Res. Mol. Brain Res. 49, 157–164. G003033/1 to A.S.I.L., J.R.E.D., and D.D.B. and by National Institutes of 19. Hurd, Y.L., Keller, E., Sotonyi, P., and Sedvall, G. (1999). Preprotachyki- Health (US) grant RO1 HD017864 to R.L.G. nin-A mRNA expression in the human and monkey brain: An in situ hybridization study. J. Comp. Neurol. 411, 56–72. Received: December 14, 2009 20. Skinner, D.C., Lang, A.L., Pahl, L., and Wang, Q. (2009). Substance Revised: February 19, 2010 P-immunoreactive cells in the ovine pars tuberalis. Neuroendocrinology Accepted: February 23, 2010 89, 3–8. Published online: April 29, 2010 21. Skinner, D.C. (2009). Rethinking the stalk effect: A new hypothesis explaining suprasellar tumor-induced hyperprolactinemia. Med. References Hypotheses 72, 309–310. 22. Hall, M.E., Miley, F., and Stewart, J.M. (1989). The role of enzymatic pro- 1. Bartness, T.J., Powers, J.B., Hastings, M.H., Bittman, E.L., and cessing in the biological actions of substance P. Peptides 10, 895–901. Goldman, B.D. (1993). The timed infusion paradigm for melatonin 23. Pennefather, J.N., Lecci, A., Candenas, M.L., Patak, E., Pinto, F.M., and delivery: What has it taught us about the melatonin signal, its reception, Maggi, C.A. (2004). Tachykinins and tachykinin receptors: A growing and the photoperiodic control of seasonal responses? J. Pineal Res. 15, family. Life Sci. 74, 1445–1463. 161–190. 24. Pisera, D., Candolfi, M., De Laurentiis, A., and Seilicovich, A. (2003). 2. Morgan, P.J., and Hazlerigg, D.G. (2008). Photoperiodic signalling Characterization of tachykinin NK2 receptor in the anterior pituitary through the melatonin receptor turns full circle. J. Neuroendocrinol. gland. Life Sci. 73, 2421–2432. 20, 820–826. 25. Wallace, J.M., Martin, G.B., and McNeilly, A.S. (1988). Changes in the 3. Hazlerigg, D., and Loudon, A. (2008). New insights into ancient seasonal secretion of LH pulses, FSH and prolactin during the preovulatory phase life timers. Curr. Biol. 18, R795–R804. of the oestrous cycle of the ewe and the influence of treatment with Encoding Long-Day Signals in the Sheep Pituitary 835

bovine follicular ﬂuid during the luteal phase. J. Endocrinol. 116, 123–135. 26. Devnath, S., and Inoue, K. (2008). An insight to pituitary folliculo-stellate cells. J. Neuroendocrinol. 20, 687–691. 27. Rebay, I., Silver, S.J., and Tootle, T.L. (2005). New vision from Eyes absent: Transcription factors as enzymes. Trends Genet. 21, 163–171. 28. Lukas, J., and Bartek, J. (2009). DNA repair: New tales of an old tail. Nature 458, 581–583. 29. Yamamura, T., Yasuo, S., Hirunagi, K., Ebihara, S., and Yoshimura, T. (2006). T(3) implantation mimics photoperiodically reduced encasement of nerve terminals by glial processes in the median eminence of Japa- nese quail. Cell Tissue Res. 324, 175–179. 30. Curlewis, J.D. (1992). Seasonal prolactin secretion and its role in seasonal reproduction: A review. Reprod. Fertil. Dev. 4, 1–23.