F-spondin Is Essential For Maintaining Circadian Rhythms
ORIGINAL RESEARCHpublished: 08 February 2018doi: 10.3389/fncir.2018.00013F-spondin Is Essential forMaintaining Circadian RhythmsGabriela L. Carrillo 1,2 , Jianmin Su 1 , Aboozar Monavarfeshani 1,3 and Michael A. Fox 1,3,4 *1Developmental and Translational Neurobiology Center, Virginia Tech Carilion Research Institute, Roanoke, VA, United States,Graduate Program in Translational Biology, Medicine and Health, Virginia Tech, Blacksburg, VA, United States, 3 Departmentof Biological Sciences, Virginia Tech, Blacksburg, VA, United States, 4 Department of Pediatrics, Virginia Tech Carilion Schoolof Medicine, Roanoke, VA, United States2Edited by:Carol Mason,Columbia University, United StatesReviewed by:Sujata Rao,Cleveland Clinic, United StatesAlexandra Rebsam,Institut National de la Santé et de laRecherche Médicale (INSERM),FranceSamer Hattar,Section on Light and CircadianRhythms (SLCR), National Institute ofMental Health, United States*Correspondence:Michael A. Foxmafox1@vtc.vt.eduReceived: 06 September 2017Accepted: 25 January 2018Published: 08 February 2018Citation:Carrillo GL, Su J, Monavarfeshani Aand Fox MA (2018) F-spondin IsEssential for Maintaining CircadianRhythms.Front. Neural Circuits 12:13.doi: 10.3389/fncir.2018.00013The suprachiasmatic nucleus (SCN) is the master pacemaker that drives circadianbehaviors. SCN neurons have intrinsic, self-sustained rhythmicity that is governed bytranscription-translation feedback loops. Intrinsic rhythms within the SCN do not matchthe day-night cycle and are therefore entrained by light-derived cues. Such cues aretransmitted to the SCN by a class of intrinsically photosensitive retinal ganglion cells(ipRGCs). In the present study, we sought to identify how axons from ipRGCs targetthe SCN. While none of the potential targeting cues identified appeared necessaryfor retinohypothalamic innervation, we unexpectedly identified a novel role for theextracellular matrix protein F-spondin in circadian behavior. In the absence of F-spondin,mice lost their ability to maintain typical intrinsic rhythmicity. Moreover, F-spondin lossresults in the displacement of vasoactive intestinal peptide (VIP)-expressing neurons,a class of neurons that are essential for maintaining rhythmicity among SCN neurons.Thus, this study highlights a novel role for F-spondin in maintaining circadian rhythms.Keywords: suprachiasmatic nucleus, extracellular matrix, circadian rhythm, photoentrainment, intrinsicallyphotosensitive retinal ganglion cellsINTRODUCTIONMost light-sensitive organisms contain biological clocks that entrain biochemical, physiologicaland behavioral processes with daily fluctuations of the solar day-night cycle. In mammals,circadian rhythmicity results from the cell-autonomous cyclic transcription and translationof tightly regulated core circadian ‘‘clock’’ genes, including period genes (per1, per2, per3),cryptochrome genes (cry1 and cry2), brain and muscle arnt-like 1 (bmal1) and circadian locomotoroutput cycles kaput (clock, Bell-Pedersen et al., 2005; Dibner et al., 2010; Mohawk et al., 2012).The cyclic expression and activity of proteins encoded by these core ‘‘clock’’ genes do notprecisely match the 24-h day-night cycle, but rather must be entrained to changes in this cycleby environmental cues. The principle cue that entrains mammalian circadian rhythms to thesolar day-night cycle is light. Environmental lighting conditions are detected and transformedinto neural activity by retinal photoreceptors and are relayed to a set of densely packed neuronsin the suprachiasmatic nucleus (SCN) of the ventral hypothalamus by retinal ganglion cells(RGCs; Figures 1A,B; Fox and Guido, 2011). The SCN then functions as the master regulatorof circadian clocks throughout the body and orchestrates the coordination of molecular andbiochemical oscillations in all other tissues (Dibner et al., 2010; Mohawk et al., 2012). Neuronswithin the SCN generate self-sustained oscillations of the core ‘‘clock’’ machinery and intercellularsignaling between these neurons, through both synaptic connections and gap junction coupling,Frontiers in Neural Circuits www.frontiersin.org1February 2018 Volume 12 Article 13
Carrillo et al.F-spondin Is Essential for Maintaining Circadian RhythmsMATERIALS AND METHODSallow for the rapid synchronization of light-induced geneexpression and oscillations (Challet et al., 2003; Aton and Herzog,2005). In the absence of light-derived signals, core ‘‘clock’’machinery within SCN neurons maintain their oscillatoryactivity but these oscillations ‘‘free-run’’ and fail to entrain tochanges in the solar day-night cycle (Foster, 1998; Brzezinskiet al., 2005).Despite the importance of light-derived signals in entrainingSCN neurons, only a small subset of RGCs supply photicinformation to the SCN (Morin and Studholme, 2014). Thissmall set of RGCs express the photopigment melanopsin andare intrinsically photosensitive (Hattar et al., 2002, 2006). Lossof these intrinsically photosensitive RGCs (ipRGCs) leads to‘‘free-running’’ circadian rhythms even in the presence of anormal solar day-night cycle (Guler et al., 2008). Despite havingbeen under extreme scrutiny since their discovery almost twodecades ago, the cellular and molecular mechanisms responsiblefor regulating ipRGC axon innervation of the SCN remainunclear (Fox and Guido, 2011). In the present study, wesought to answer this question by identifying and testingSCN-specific axonal targeting cues. Using a bio-informaticsapproach, we identified F-spondin and Slit1, two extracellularmatrix proteins, and ALCAM, a cell adhesion molecule, thatwere all enriched in the adult SCN. The ability of thesethree cues to direct axonal growth, guidance and targetingin other regions of the developing brain has been wellestablished (Ott et al., 1998; Burstyn-Cohen et al., 1999;Tzarfati-Majar et al., 2001; Plump et al., 2002; Diekmannand Stuermer, 2009). In fact, two of these cues (Slit1 andALCAM) have established roles in the development of theretinofugal pathway in rodents (Ott et al., 1998; Weiner et al.,2004; Buhusi et al., 2009; Diekmann and Stuermer, 2009).While F-spondin acts as a guidance cue in developing motorcircuits (Burstyn-Cohen et al., 1999; Tzarfati-Majar et al., 2001),its role in the developing visual system remains unexplored.It is noteworthy, however, that F-spondin shares homologywith the extracellular matrix protein Reelin and can bindcanonical Reelin receptors, both of which are important for theassembly of connections between ipRGCs and visual thalamus(Tzarfati-Majar et al., 2001; Hoe et al., 2005; Su et al., 2011,2013).Here, we tested whether these factors were necessaryfor the formation of connections between ipRGCs and theSCN. While our results demonstrate that each of thesecues is dispensable for retinohypothalamic targeting, wefound that F-spondin-deficient (spon1 / ) mutants (but notmutants lacking Slit1 or ALCAM) displayed severely disrupted‘‘free-running’’ rhythmicity. Furthermore, F-spondin-deficientmutants displayed overtly normal circadian rhythms in normalday-night conditions, suggesting that the loss of intrinsiccircadian rhythmicity was masked in the presence of light.The rapid ability of spon1 / mutants to adapt to changes inlighting conditions and to entrain to ultradian photoperiodssuggests that SCN neurons are weakly coupled in the absenceof this extracellular matrix protein. Taken together, these resultsidentify a novel role for F-spondin in maintaining intrinsiccircadian rhythms.Frontiers in Neural Circuits www.frontiersin.orgAnimalsC57BL/6 mice were obtained from Charles River Laboratories(Wilmington, MA, USA). Spon1 / mutant mice werepurchased from Taconic Biosciences Inc. (Hudson, NY, USA)and slit1 / were purchased from MMRRC1 . The generationof alcam / , math5 / and opn4taulacz/ mice were describedpreviously (Wang et al., 2001; Weiner et al., 2004; Hattar et al.,2006). Genomic DNA was isolated from tail using the HotSHOTmethod (Truett et al., 2000) and genotyping was performedwith the following primers: spon1 (wildtype, WT) 50 -GAC CGGAGA TCT AGG AAC CCC TAG-30 and 50 -CAC TCT CGCCAA CAG CTG GAG CG-30 , spon1 (mutant) 50 -CTC CGC TCAGAG CAG CGC AGC TC-30 and 50 -CCC TAG GAA TGC TCGTCA AGA-30 ; lacZ 50 -TTC ACT GGC CGT CGT TTT ACAACGTCG TGA-30 and 50 -ATG TGA GCG AGT AAC AAC CCGTCG GAT TCT-30 ; math5, ATG GCG CTC AGC TAC ATCAT and GGG TCT ACC TGG AGC CTA GC; neomycin (neo),GCC GGC CAC AGT CGA TGA ATC and CAT TGA ACAAGA TGG ATT GCA; slit1 (WT) 50 -AAG ATG CCT CCT CTGACT TC-30 and 50 -ACC CTT AGC TTC TAC CAA CC-30 ; slit1(mutant) 50 -TCT CCT TTG ATC TGA GAC CG-30 and 50 -AGGTTT CTC GAG CGT CAT AG-30 ; alcam (common) 50 -AAAGTC GCT GTC CCC CTA AG-30 , alcam (mutant) 50 -GGT CTTGTA GTT GCC GTC GT-30 and alcam (WT) 50 -GAG CAGACC AGT CAA GCC TAA-30 . The following cycling conditionswere used on an Eppendorf or Bio-Rad Mastercycler EP: spon1,94 C for 5 min, followed by 33 cycles of amplification (94 C for30 s, 62 C for 30 s, and 72 C for 45 s) and 10 min at 72 C; lacZ,95 C for 5 min, followed by 35 cycles of amplification (95 C for30 s, 52 C for 30 s, 72 C for 45 s), and 10 min at 72 C; math5,95 C for 5 min, followed by 35 cycles of amplification (94 C for30 s, 59 C for 30 s, and 72 C for 45 s) and 10 min at 72 C; neo,94 C for 3 min, followed by 35 cycles of amplification (94 C for30 s, 56 C for 30 s, and 72 C for 45 s) and 10 min at 72 C; slit1,95 C for 5 min, followed by 30 cycles of amplification (95 Cfor 30 s, 60 C for 30 s, and 72 C for 30 s) and 10 min at 72 C;alcam, 94 C for 2 min, followed by 10 cycles of amplification(94 C for 20 s, 65 C for 15 s and decrease 0.5 C per cycle, 68 Cfor 10 s) and additional 28 cycles of amplification (94 C for 15 s,60 C for 15 s, and 72 C for 10 s) and 1 min at 72 C. All analysesconformed to National Institutes of Health (NIH) guidelines andprotocols, approved by the Virginia Polytechnic Institute andState University Institutional Animal Care and Use Committees.ReagentsAll chemicals and reagents were purchased from Fisher(Fairlawn, NJ, USA) or Sigma (St. Louis, MO, USA) unlessotherwise stated.ImmunohistochemistryFluorescent immunohistochemistry (IHC) was performed on16 µm cryosectioned paraformaldehyde (PFA)-fixed brain tissueas described previously (Su et al., 2010, 2012). Briefly, tissue slides1 https://www.mmrrc.org2February 2018 Volume 12 Article 13
Carrillo et al.F-spondin Is Essential for Maintaining Circadian RhythmsZ1 LSM 710 confocal microscope or a Zeiss LSM 700 confocalmicroscope.were allowed to air dry for 15 min before being incubated withblocking buffer (2.5% normal goat serum, 2.5% bovine serumalbumin and 0.1% Triton X-100 in PBS) for 30 min. Rabbitanti-Vasoactive Intestinal Peptide (VIP) antibodies (diluted1:150 for IHC) were purchased from Immunostar and rabbitanti-vasopressin (AVP) antibodies (diluted 1:1000 for IHC)were purchased from Millipore (Cat# AB1565). Fluorescentsecondary antibodies were from Life Technologies (diluted1:1000). Primary antibodies were diluted in blocking bufferand incubated on tissue sections for overnight at 4 C. On thefollowing day, tissue slides were washed in PBS and secondaryantibodies diluted 1:1000 in blocking buffer were applied toslides for 1 h at room temperature. After thoroughly washingin PBS, tissue slides were coverslipped with VectaShield (VectorLaboratories, Burlingame, CA, USA). Images were acquired on aZeiss Axio Imager A2 fluorescent microscope, a Zeiss ExaminerZ1 LSM 710 confocal microscope, or a Zeiss LSM 700 confocalmicroscope (Oberkochen, Germany). Intensity and area of signaloccupation of fluorescent, confocal images were measured inImageJ as previously described (Singh et al., 2012; Su et al., 2016).A total of 4–6 animals (three sections per animal) were analyzedper genotype and Student’s T tests were used to assess statisticalsignificance.Quantitative Real-Time PCRRNA was isolated from the SCN from both 12:12 LD andDD conditions. Samples from 12:12 light:dark (LD) wereisolated at ZT4, ZT10, ZT16 and ZT22. Samples from constantdarkness (DD) conditions were isolated at ZT4 5 days aftermice were exposed to constant darkness. For samples obtainedfrom the dark phase of ther standard 12:12 LD cycle or fromanytime during a DD cycle, mice were euthanized and brainremoved in the dark under dim red light illumination. In allcases, brains were removed, vibratomed (200 µm) in iced-coldDEPC-PBS and Suprachiasmatic nuclei (SCN) were dissected.For each sample (at each time point) SCN from a total ofsix mice was pooled. RNA from these SCN was isolated usingthe BioRad Total RNA Extraction from Fibrous and FattyTissue kit (BioRad). cDNAs were generated from 200 ng RNAwith the Superscript II Reverse Transcription First StrandcDNA Synthesis kit (Invitrogen). Quantitative real-time PCR(qPCR) was performed on a Chromo4 Four Color Real-Timesystem (BioRad) using iQ SYBRGreen Supermix (BioRad;Su et al., 2010). qPCR was performed with the followingprimers Bmal1: 50 -TCCTTCCAGGCAGTCAACTT-30 and50 -CTGCAGTGAATGCTTTTGGA-30 ; cry1: 50 -TGG CATCAA GAT CCT CAA GA-30 and 50 -TCC GCT GCG TCTATA TCC TC-30 ; gjd2: 50 -TGCTCATCATCGTACACCGT30 and 50 -GCAGCAGCACTCCACTATGA-30 ; gapdh: 50 -CGTCCCGTAGACAAAATGGT-30 and 50 -TTGATGGCAACAATCTCCAC-30 ; per1: 50 -AAC GCT TTG CTT TAG ATC GG-30and 50 -TCC TCA ACC GCT TCA GAG AT-30 ; per2: 50 -GTATCC ATT CAT GTC GGG CT-30 and 50 -TAC TGG GAC TAGCGG CTC C-30 ; vip: 50 -CGT GGT TGT TTT CCT TCG AG-30and 50 -GGA GCA GTG AGG GAG ATT CTG-30 . qPCR primerswere designed over introns. The following cycling conditionswere used with 10 ng RNA: 95 C for 30 s, followed by 40 cyclesof amplification (95 C for 5 s, 60 C for 30 s, 55 C for 60 s, readplate) and a melting curve analysis. Relative quantities of RNAwere determined using the CT method. A minimum ofn 3 experiments (each in triplicate) was run and examined forspon1 / mutants and littermate controls at each time point(ZT4, ZT10, ZT16, ZT22) during 12:12 LD or DD (ZT4). Eachindividual run included separate gapdh control reactions at eachtime point.In Situ HybridizationIn situ hybridization (ISH) was performed on 16 µm coronalcryosectioned tissues as previously described. Antisenseriboprobes were generated from full length cDNAs of slit1(MMM1013-98685876), alcam (MMM1013-202762192), spon1(MMM1013-202701079) and per1 (MMM1013-202764685;from Open Biosystems; Huntsville, AL, USA). Riboprobeswere synthesized using digoxigenin (DIG)-labeled UTP(Roche, Mannheim, Germany) and the MAXIscript in vitroTranscription Kit (Ambion, Austin, TX, USA). Probes werehydrolyzed to 500 nt. Coronal brain sections were prepared andhybridized at 65 C as previously described (Su et al., 2010), andbound riboprobes were detected by horseradish peroxidase(POD)-conjugated anti-DIG antibodies and fluorescentstaining with Tyramide Signal Amplification (TSA) systems(PerkinElmer, Shelton, CT, USA). Images were obtained ona Zeiss Axio Imager A2 fluorescent microscope or a ZeissExaminer Z1 LSM 700 confocal microscope. A minimum ofthree animals per genotype, age and time were compared in ISHexperiments.Intraocular Injection of AnterogradeTracersWheel Running Activity AssayWheel running activity was monitored in wheel cages fromLafayette Instruments which recorded each wheel revolution asan event and sent that information to a compatible computerin 5-min bins using ClockLab software R2011b. All mice wereindividually housed. Mice were habituated for 2 days beforecontinually recording activity for 2 weeks of 12:12 LD and2 weeks of DD. To test phase advance and phase delay, activitywas recorded for 2 weeks in 12:12 LD, then the onset of lightwas advanced 6 h and activity was recorded in 12:12 LD. After2 weeks, the onset of light was delayed 6 h to the original onsetof light for 2 weeks of LD. A total of six spon1-mutant andIntraocular injection of cholera toxin subunit B (CTB)conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen)was performed in P13 mice as described previously (Su et al.,2011, 2013). After 2 days, mice were killed, and brains werefixed in 4% PFA. One hundred micrometer coronal sectionswere sectioned on a vibratome (Microm HM 650 V; ThermoScientific, Waltham, MA, USA) and mounted in VectaShield(Vector Laboratories, Burlingame, CA, USA). Retinal projectionsin SCN were analyzed from at least three animals for eachage and genotype. Images were acquired on a Zeiss ExaminerFrontiers in Neural Circuits www.frontiersin.org3February 2018 Volume 12 Article 13
Carrillo et al.F-spondin Is Essential for Maintaining Circadian Rhythmssix littermate WT mice were analyzed for the phase advanceand delay conditions. To test activity in skeleton photoperiods,spon1 / and littermate controls were exposed to 2 weeks of12:12 LD then were exposed to 2 weeks of 1:11 LD cycle andfinally 2 weeks of 12:12 LD. To assess spon1 / and littermatecontrol mice to an ultradian solar cycle, mice were exposed tomore than 2 weeks of 12:12 LD circle, then were exposed to1 week of 3.5 h light/3.5 h dark.retinohypothalamic innervation in targeted mouse mutantslacking each cue (i.e., slit1 / , alcam / and spon1 / mutants). All three mutants are viable, fertile, and appearindistinguishable from littermate controls in regards to size andcage activity. To assess retinohypothalamic innervation in thesemutants, we performed intraocular injections of fluorescentlyconjugated CTB, an anterograde tracer that efficiently labelsall retinal projections into the brain (Muscat et al., 2003). Incontrols, retinal projections from each eye project bilaterallyto the SCN in each hemisphere on the hypothalamus andthese projections arborize to fill the entire SCN (Figure 2A).To our surprise CTB-labeled retinohypothalamic projectionsappear unaltered by the loss of Slit1, ALCAM or F-spondin(Figures 2B–D).Since CTB labels all retinal projections indiscriminately,despite only a very small population of RGCs actually projectingaxons to the SCN, it remained possible that axons from ipRGCswere no longer innervating SCN in these mutants and insteadother classes of RGC axons were present. Consequently, wetested whether ipRGC axons correctly innervated the SCNin mutants lacking F-spondin, the targeting candidate whosedevelopmental expression pattern most closely resembled thedistribution of ipRGC arbors in SCN (Figure 1D; Hattar et al.,2006). Spon1 / mutant mice were crossed with opn4tau-lacz/ transgenic mice, in which ipRGC projections are labeledwith Tau-LacZ fusion protein (Hattar et al., 2002, 2006).The majority of retinal projections in the SCN of controlopn4tau-laz/ mice contained Tau-LacZ (Figure 2E). Patternsof ipRGC axon arbors in mutants were indistinguishablefrom controls (Figure 2F). Altogether, our results stronglysuggest Slit1, ALCAM and F-spondin are each individuallydispensable for the formation of the retinohypothalamictract.RESULTSSlit1, ALCAM, F-spondin Are Enriched inthe Developing Suprachiasmatic NucleusTo begin to identify potential retinohypothalamic targetingcues we screened available on-line expression databases for anyextracellular matrix proteins, growth factors, morphogens orcell adhesion molecules enriched in adult mouse SCN (Leinet al., 20072 ). We identified two genes encoding extracellularmatrix proteins (Slit1 and F-spondin) and one gene encodinga transmembrane cell adhesion molecule (ALCAM) all ofwhich are enriched in adult SCN. Two of these candidateshave previously been reported to contribute to the guidanceand growth of retinal axons: members of the Slit family ofextracellular matrix proteins mediate retinal pathfinding throughbinding to Robo receptors (Ringstedt et al., 2000; Plumpet al., 2002; Thompson et al., 2006) and the immunoglobulinsuperfamily adhesion molecule ALCAM (also called BEN, SC1,DM-GRASP, Neurolin and CD166) contributes to the guidance,fasciculation, and topographic mapping of retinal axons (Ottet al., 1998; Weiner et al., 2004
biochemical oscillations in all other tissues (Dibner et al.,2010;Mohawk et al.,2012). Neurons within the SCN generate self-sustained oscillations of the core ‘‘clock’’ machinery and intercellular signaling between these neurons, thr
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