A Neural Basis For Control Of Cichlid Female Reproductive .
ReportA Neural Basis for Control of Cichlid FemaleReproductive Behavior by Prostaglandin F2aHighlightsdProstaglandin F2a injection rapidly leads to naturalistic femalespawning behaviordA single receptor for prostaglandin F2a, Ptgfr, is expressed infour brain regionsdDeletion of Ptgfr with CRISPR yields females that do notexhibit sexual behaviorAuthorsScott A. Juntti, Austin T. Hilliard,Kai R. Kent, ., Jasmine L. Loveland,Philippe Mourrain, Russell D. FernaldCorrespondencesjuntti@stanford.eduIn BriefJuntti et al. use CRISPR/Cas9 geneediting and pharmacology to show thatprostaglandin F2a signaling is necessaryand sufficient for female sexual behaviorin a cichlid fish. They identify a receptorfor prostaglandin F2a and implicatespecific brain regions in sexual behavior.Juntti et al., 2016, Current Biology 26, 943–949April 4, 2016 ª2016 Elsevier Ltd All rights 7
Current BiologyReportA Neural Basis for Control of Cichlid FemaleReproductive Behavior by Prostaglandin F2aScott A. Juntti,1,* Austin T. Hilliard,1 Kai R. Kent,1 Anusha Kumar,1 Andrew Nguyen,1 Mariana A. Jimenez,1Jasmine L. Loveland,1 Philippe Mourrain,2 and Russell D. Fernald11Departmentof Biology, Stanford University, Stanford, CA 94305, USAof Psychiatry and Behavioral Sciences, Center for Sleep Sciences, Stanford University, Stanford, CA 94305, USA*Correspondence: b.2016.01.0672DepartmentSUMMARYIn most species, females time reproduction to coincide with fertility. Thus, identifying factors that signalfertility to the brain can provide access to neural circuits that control sexual behaviors. In vertebrates,levels of key signaling molecules rise at the time offertility to prime the brain for reproductive behavior[1–11], but how and where they regulate neural circuits is not known [12, 13]. Specifically, 17a,20b-dihydroxyprogesterone (DHP) and prostaglandin F2a(PGF2a) levels rise in teleost fish around the time ofovulation [10, 14, 15]. In an African cichlid fish, Astatotilapia burtoni, fertile females select a mate andperform a stereotyped spawning routine, offeringquantifiable behavioral outputs of neural circuits.We show that, within minutes, PGF2a injectionactivates a naturalistic pattern of sexual behaviorin female A. burtoni. We also identify cells in thebrain that transduce the prostaglandin signal tomate and show that the gonadal steroid DHP modulates mRNA levels of the putative receptor forPGF2a (Ptgfr). We use CRISPR/Cas9 to generatethe first targeted gene mutation in A. burtoni andshow that Ptgfr is necessary for the initiation of sexual behavior, uncoupling sexual behavior from reproductive status. Our findings are consistent with amodel in which PGF2a communicates fertility statusvia Ptgfr to circuits in the brain that drive femalesexual behavior. Our targeted genome modificationin a cichlid fish shows that dissection of gene function can reveal basic control mechanisms for behaviors in this large family of species with diverse andfascinating social systems [16, 17].RESULTSWe sought to understand the control of the complex spawningbehavioral routine in a cichlid fish, A. burtoni. In this species,the male dramatically displays his body coloration to a femalewhile quivering vigorously and then attempts to lead her backto his territory (Figure 1A; Movie S1). If the female is ready tospawn, she follows him into his spawning site and pecks ategg-like spots on the anal fin of the male as he quivers in frontof her. She then lays eggs and immediately collects them fromthe substrate into her mouth. As she searches for more eggs,she pecks again at egg spots near the site of sperm releasefrom the male, fertilizing the eggs. The male and female circlearound one another several times, repeating these behaviors insequence. The female then carries the embryos in her mouthfor !2 weeks as they develop.We first asked whether this spawning routine could be elicitedby PGF2a, a factor whose titers rise in the fertile female fish [18].Within 30 min of intraperitoneal PGF2a injection into femalesvisually identified as non-fertile, they exhibited behavior quitesimilar to natural spawning behavior, whereas vehicle-injectedcontrols rarely showed the full sequelae of reproductive behaviors (PGF2a-injected, 43% circled; vehicle-treated, 7% circled;n 28/group; p 0.0043; Fisher’s exact test; Figure S1A; MovieS2). Females performed all behavioral sequences typical ofreproductive behavior except egg laying, because we selectedonly fish that had not ovulated. Differences between PGF2aand vehicle-injected fish cannot be ascribed to differences inmale behavior or reproductive stage (assessed by ovary mass),as these parameters did not differ between assays with PGF2aand vehicle-injected females (Figures 1B, 1C, 1G, and 1H).Female following and spawning site entry was not significantlyincreased (Figures 1D and 1E), implying that PGF2a promotesthe final stage of spawning behavior, circling (Figure 1F). Wecompared the behavior of naturally spawning females to a subset of PGF2a-injected females that exhibited comparable levelsof reproductive behaviors by testing the frequency of transitionsbetween behaviors. We found that PGF2a-injected females weresimilar to naturally spawning females in frequency and orderingof reproductive behaviors (Figure 1J). Thus, the behavioralsequelae elicited by PGF2a are very similar to those of naturallyspawning females but are performed outside the time of fertility.This rapid generation of a complex behavior that we andothers [8, 11] observe in cichlids led us to seek the mechanismof action for PGF2a at the genetic and neural levels. We identified a family of 11 putative G protein-coupled receptors forprostaglandins in the A. burtoni genome and show that only asingle receptor forms a monophyletic clade with PGF2a receptors (Ptgfr) from other vertebrate genomes (Figure S2A). ThisA. burtoni G protein-coupled receptor has conserved residuesfor PGF2a signaling, and elements of synteny are maintainedfrom cichlid to human (Figures S2B and S2C), suggesting thatit is the sole Ptgfr ortholog, and has maintained PGF2a signalingcapability.Current Biology 26, 943–949, April 4, 2016 ª2016 Elsevier Ltd All rights reserved 943
Figure 1. PGF2a Activates Female Reproductive Behavior in A. burtoni(A) Natural progression of spawning behavior. After ovulation, females lay eggs during circling.(B–I) Analysis of all reproductive assays. Males quiver, lead, and attack similarly with vehicle and PGF2a-injected females (B, C, and G). Vehicle- and PGF2ainjected females show similar following and pot entry (D and E), but females show circling behavior more rapidly and frequently after PGF2a injection (F and I).Body-weight-normalized ovary mass does not differ between groups (H). *p 0.0018, Mantel-Cox test; **p 0.0010, Mann-Whitney U test. Mean SEM; n 28females per treatment.(J) Ethograms reveal that naturally spawning and a reproductive subset of PGF2a-injected females exhibit quantitatively similar transitions between reproductivebehaviors. Vehicle-treated females were matched for normalized ovary mass but rarely circle. Diameters of circles are proportional to count of each behavior;weights of arrows are proportional to fraction followed by second behavior. Transitional probabilities do not differ between naturally spawning and PGF2a-injectedfemales; all transitions p 0.05, Mann-Whitney U test. n 5 assays per group.See also Figure S1 and Movies S1 and S2.A prior study in goldfish found that PGF2a injection directlyinto the brain is more potent than systemic injection for eliciting reproductive behavior and that ovariectomized femalesspawn in response to PGF2a [19]. Together, these resultsindicate that PGF2a acts on target(s) in the brain. Therefore,we localized cells expressing Ptgfr in the brain using in situhybridization (ISH) and found expression in only four regions(Figures 2A, 2B, and S3A–S3D): the preoptic area (POA), aregion implicated in sexual behavior across vertebrates [20];the lateral tubular nucleus (NLT), a suggested homolog of themammalian arcuate nucleus [21]; the vagal lobe (VL), a regionthat communicates with the internal viscera and controlsmouth movements [22] but has not previously been implicatedin female reproduction; and the dorsal compartment of theventral telencephalon (Vd), a subpallial structure with noknown function [23].Because individual neurons activated during female spawningbehavior have not been previously identified, we asked whetherthese brain regions were active during spawning by using cFosmRNA expression, a proxy for recent neural activity. We alloweduninjected females to interact with a courting male andcompared cFos expression in females that spawned to thosethat did not. Cells in the POA and VL exhibited greater cFosexpression in females that had spawned naturally when exposedto males compared to females that did not spawn (POA, 2.4-foldincrease in spawning females, p 0.012, Mann-Whitney test; VL,only had cFos expression in spawners; n R 7/group; Figure 2).NLT was cFos in females exposed to males regardless of944 Current Biology 26, 943–949, April 4, 2016 ª2016 Elsevier Ltd All rights reserved
Figure 2. Ptgfr Is Expressed in RegionsActive during Spawning and Rises at theTime of Spawning(A and B) Ptgfr mRNA is expressed in the POA andin scattered cells of the VL.(C–F) POA and VL express cFos mRNA after beingallowed to spawn naturally when exposed to amale (C and D). Females exposed to a courtingmale that did not spawn did not show cFosexpression (E and F). The scale bars represent100 mm; n 6–11 per group.See also Figures S2 and S3 and Table S1.spawning behavior (Figures S3I–S3L), and we did not detect arobust cFos induction in Vd in any fish (data not shown). Takentogether, these results indicate that Ptgfr regions POA and VLplay a role in reproductive behavior, whereas Ptgfr in Vd andNLT likely are involved in non-spawning behaviors.Ptgfr expression varied systematically across the reproductivecycle, with Ptgfr mRNA staining increased !3-fold in the POAof females that had just spawned naturally, relative to femaleswith small or large ovaries earlier in the reproductive cycle(Figures 3A–3D), indicating that its expression is enhanced onlybriefly around the time of spawning. The teleost progestin,17a,20b-dihydroxyprogesterone (DHP), promotes final maturation and ovulation of oocytes prior to spawning in most teleosts including cichlids [15, 24, 25]. We found that the progesterone receptor (Pgr) is present in the female POA (Figure 3E). Totest the role of this signaling pathway in Ptgfr expression, wetreated ovariectomized females with either DHP or vehicle andmeasured Ptgfr mRNA in the POA after 3 hr. DHP treatmentcaused an increase in Ptgfr mRNA in the POA similar to thatobserved in naturally spawning females (Figures 3F–3H), suggesting that Pgr signaling at ovulation increases sensitivity toPGF2a, promoting spawning behavior.To directly test the role of Ptgfr in spawning behavior, wegenerated A. burtoni carrying mutant Ptgfr using the CRISPR/Cas9 system [26]. We injected single-cell embryos with Cas9mRNA [27, 28] and a single-guide RNA targeting the secondtransmembrane domain of Ptgfr (Figures 4A and 4B). We raisedinjected embryos to adulthood, separated them by sex, andhoused them with wild-type fish of the opposite sex. We foundthat none of the injected female G0 fish produced offspring(n 9 females). When we sequenced fin clip genomic DNAfrom injected females, we found that CRISPR/Cas9 is efficientin A. burtoni: sequencing of genomic DNA from 20/22 (91%) injected fish exhibited extensive modification of the Ptgfr locus(Figure S4). Thus, CRISPR/Cas9 can be used to cause indelsin A. burtoni, enabling the rapid phenotyping of mutant cichlids.CRISPR/Cas9 has been suggested to cause low rates of offtarget mutations [28], so we outcrossed G0 males to wild-typefemales to isolate the effect of targeted mutations. Ptgfr mutantmales produced numerous broods with wild-type females. Thiscontrast with Ptgfr mutant females suggested a sex-specificrole for Ptgfr in spawning. Half of G1offspring (49%) carried Ptgfr indel alleles,indicating numerous mutant cells in thegermline (n 92 fish). These G1 offspringcarried a variety of indel alleles, so we selectively propagatedthose predicted to result in a loss of function (i.e., two frameshiftmutations and a 177-bp deletion). We intercrossed G1 fish fromdifferent founders to obtain biallelic Ptgfr mutants (PtgfrD/D).These crosses transmitted modified Ptgfr alleles at expectedfrequencies, and PtgfrD/D fish are viable (Table S1). To assesswhether PtgfrD/D females would reproduce naturally, wecollected fin clips from females observed carrying broods.PCR amplification and sequencing revealed that no mouthbrooding females were PtgfrD/D (Figure 4I).Although these results indicate that Ptgfr is critical for reproduction, we asked whether Ptgfr mutant females would performcomplete spawning behavior routines. We injected these females and their wild-type siblings with PGF2a and paired eachwith a wild-type singly housed male. PtgfrD/D females never exhibited the circling behavior typical of their wild-type siblings(p 0.04; Fisher’s exact test; n 8–13/genotype), though theydid perform the initial components of the routine (Figure 4D).These phenotypes were not a result of differential courtship bymales or female size, and ovary mass indicated females hadhigh GSI (gonadosomatic index; Figures 4E–4H). Thus, PGF2asignaling through Ptgfr is necessary for the final stages of reproductive behavior, and there appears to be no redundancy insignaling pathways that activate spawning.DISCUSSIONOur results show that PGF2a signaling is necessary and sufficientto induce the final stages of reproductive behavior in cichlid fish,and we have identified regions in the brain likely important forgenerating this behavior. Although the neural circuit for spawning requires Ptgfr, other factors must modulate its action. Forexample, we found that PGF2a robustly activated spawningbehavior in 8/10 wild-type females with larger ovaries (gonadosomatic index 1.4) but 0/7 females with smaller ovaries (Figure S1B), suggesting that some factor(s) other than DHP inhibitssexual behavior after recent egg laying. PGF2a-insensitive periods have been observed later in the reproductive cycle in theparadise fish [10], though PGF2a appears uniformly effective inother species [11, 29]. Additionally, Ptgfr mRNA levels in thePOA rise in A. burtoni during a separate short time windowCurrent Biology 26, 943–949, April 4, 2016 ª2016 Elsevier Ltd All rights reserved 945
Figure 3. Progestin Signaling Upregulates Ptgfr mRNA Expression in POA(A–D) Ptgfr mRNA levels rise in the POA around the time of spawning. Females with small (A) or large (B) ovary mass but that did not spawn had less Ptgfr mRNAexpression than females that spawned 30 min prior (C). *p 0.0009 by Kruskal-Wallis test and p 0.05 by Dunn’s post hoc test; n 6–11 females per group.(E) Progesterone receptor is expressed in the Ptgfr compartment of the POA.(F–H) Treatment of ovariectomized (OVX) females with 17a,20b-dihydroxyprogesterone (DHP) results in a rise in Ptgfr mRNA levels. *p 0.0286 by Mann-Whitneytest; n 4 females/group.Mean SEM; scale bar, 100 mm.around spawning, suggesting that Ptgfr neurons become moresensitive to PGF2a when females are fertile.We propose that individual Ptgfr regions control discreteaspects of female reproduction. The POA has been implicatedin sexual behavior across vertebrates [20, 30, 31], and theexpression of Ptgfr mRNA in this region is consistent with arole in spawning behavior. Given the known roles of the VL, PtgfrmRNA expression here may identify neurons that are importantfor the initiation of mouthbrooding or for receiving signals fromviscera including the reproductive tract. Accordingly, femalesthat exhibit circling behavior subsequent to PGF2a injection donot show cFos mRNA in the VL (data not shown), implying thategg laying and/or mouthbrooding is controlled by these neurons.Are these regions activated simultaneously to drive reproductivebehavior, or might one region be the site at which the PGF2asignal from the periphery acts? Cells in the POA could be accessible to circulating prostaglandins, similar to mammals [32]. In analternative model, fertility signals from the reproductive tractcould be communicated to the brain via another signal, triggeringthe neural synthesis of PGF2a. One candidate for such a mediator is the vagus nerve, which innervates the viscera and communicates with the VL. A comparison of Ptgfr expressionpatterns across brain regions in species with divergent behavioral patterns may highlight the presence or absence of Ptgfr neuronal subsets, allowing inferences about specific functionsfor individual populations. The development of CRISPR/Cas9in cichlids will allow direct tests of such hypotheses using celltype-specific gene modifications.In mammals, PGF2a promotes both the onset of labor andmaternal behavior [2, 6]. Our data, taken together with resultsfrom other vertebrates including ovoviviparous fish [33], suggests that PGF2a signaling has an ancestral function linking therelease of offspring or eggs from the reproductive tract withthe appropriate behavior. In mammals, for which sexual behavioris temporally dissociated from parturition, either progesteroneor prostaglandin E2 (PGE2) is sufficient to drive female sexualbehavior after priming by estradiol [1, 4, 34, 35]. PGE2 signalingtherefore may act in a signaling pathway that anticipates ovulation in order to time sexual behavior with fertility [29]. This function could result from a gain of expression of a PGE2 receptor inan ancestral mating circuit or from co-opting of PGE2-sensitivecells into a mating circuit. Future experiments may reveal aconserved pattern of gene expression in cells that regulate mating in mammals [36, 37] and fish.Given the remarkable diversity in reproductive behaviorsamong the !1,500 known cichlid species [17], genetically modified cichlids [38] have the potential to test the function of specificgenes, neurons, and hormones that control social behaviors andto reveal their evolutionary trajectories. Furthermore, becausethe prolific speciation of East African cichlids is postulatedto result from sexual selection [39], Ptgfr cells are a crucialcomponent of a circuit through which females select a partner946 Current Biology 26, 943–949, April 4, 2016 ª2016 Elsevier Ltd All rights reserved
Figure 4. CRISPR/Cas9-Mediated Mutation of Ptgfr Results in Failure to Spawn(A) Schematic for generation of biallelic Ptgfr mutants. sgRNA, single-guide RNA.(B) Three Ptgfr alleles encoding a large deletion or frameshift mutations were analyzed in F1 females. The protospacer-adjacent motif is underlined and Cas9 cutsite indicated by arrowhead.(C and D) PtgfrD/D females did not initiate circling behavior in response to PGF2a injection; *p 0.031; Mantel-Cox test; n 8–13 females/genotype.(E) Ovary size was not different from wild-type (WT) males.(F–H) Males did not show different levels of courtship (F and G) or aggression (H) toward PtgfrD/D females. Mean SEM.(I) Group-housed PtgfrD/D females were not found to carry offspring despite comprising 42% of the population. **p 0.0001; Fisher’s exact test; n 93 grouphoused fish; n 31 mouthbrooders.See also Figure S4 and Table S2.and initiate mating. Thus, mapping the inputs and outputs ofthese cells will permit an understanding of how females selecta mate and ultimately shape evolution.EXPERIMENTAL PROCEDURESFish were bred and used at Stanford University from a colony derived fromLake Tanganyika [16] in accordance with AAALAC standards.Non-gravid females were identified by absence of abdominal distension dueto ovary size and injected intraperitoneally with PGF2a (!1.5 mg per g body;Cayman) or vehicle. Females were introduced into a male’s tank immediatelyafter injection. Behaviors were video recorded for 30 min. Naturally spawningfemales in Figures 2 and S3 were collected by allowing a male access to uninjected females. Brains were dissected 30 min after observing spawning andsimultaneously from a control female from the same tank that did not spawn.Behavior assays were coded by an observer blind to treatment using customMATLAB software [40].Adult female A. burtoni were allowed to recover for 1 or 2 weeks after ovariectomy (OVX) and then were injected intraperitoneally with DHP (125 ng/gbody weight; Sigma-Aldrich) or DMSO/saline vehicle. Brains were collectedat 3 hr post-injection.Raster plots and transitional probabilities were generated using a customsoftware package in R (http://fernaldlab.stanford.edu/resources). We usedMann-Whitney U tests for two-group comparisons of continuous data, Fisher’sexact test for categorical data, and Mantel-Cox test for latency data. Transitional probabilities were calculated by dividing the total number of eachbehavior by the number of instances in which the subsequent behavioroccurred. Arrow weights in Figure 1J are only shown for transitions with probability R4%. We selected five PGF2a-injected females with a similar number ofcircling bouts to compare with five naturally spawning females and matchedfive vehicle-injected females by their comparable GSI. We used Mann-WhitneyU tests to compare transition probabilities across groups, with a Bonferronicorrected cutoff of a 0.0027 to correct for 19 transitions we observed inFigure 1J.For ISH, we subcloned Ptgfr and cFos (NM 001286320), into pCR-TOPO4(Life Technologies) using the following primers: Ptgfr forward, 50 -AACCAAAGACTGGCTGGATG-30 ; Ptgfr reverse, 50 -AAATTTCGAGCCACAACAGC-30 ;cFos forward, 50 -AATTGGATCCAAGCCCAGATCTTCAGTGG-30 ; and cFosreverse, 50 -AATTGAATTCATAGCCCTGTGATCGGCAC-30 .Mutations of Ptgfr were induced by injection of a single-guide RNA (sgRNA)targeting the second transmembrane domain. We annealed oligonucleotidesgPtgfrF, 50 -TAGGCTTGAGCCCCTTGTTCCT-30 , and gPtgfrR, 50 -AAACAGGAACAAGGGGCTCAAG-30 , and ligated the product into pT7-gRNA [27, 28].We waited for 30 min of fertilization and then injected single-cell embryos.We delivered !1 nl of 12 ng/ml Ptgfr sgRNA, 60 ng/ml nls-zCas9-nls mRNA,and 0.3% Texas-Red-conjugated dextran (3,000 MW; Life Technologies).In !5-week embryos, we PCR amplified a 554-bp amplicon spanningCurrent Biology 26, 943–949, April 4, 2016 ª2016 Elsevier Ltd All rights reserved 947
the sgRNA-binding site with the primers PtgfrFlankF, 50 -CTTCTCCAACAGCCTTGCTC-30 and PtgfrFlankR, 50 -CACAGCCTGTTAGCGTGTTG-30 ,and Sanger sequenced the product with PtgfrFlankF (ElimBio). We saved fishshowing evidence of mutant Ptgfr and crossed these fish to wild-types. G1fish carrying an indel predicted to result in a null mutation were intercrossed togenerate F1 fish. Additional information can be found in the Supplemental Experimental Procedures.SUPPLEMENTAL INFORMATIONSupplemental Information includes Supplemental Experimental Procedures,four figures, two tables, and two movies and can be found with this articleonline at http://dx.doi.org/10.1016/j.cub.2016.01.067.AUTHOR CONTRIBUTIONSConceptualization, S.A.J. and R.D.F.; Methodology, S.A.J. and A.T.H.; Investigation, S.A.J., A.T.H., K.R.K., A.K., A.N., M.A.J., and P.M.; CRISPR/Cas9genome editing, S.A.J. and P.M.; Writing – Original Draft, S.A.J.; Writing – Review & Editing, S.A.J., A.T.H., P.M., and R.D.F.; Funding Acquisition, S.A.J.,P.M., and R.D.F.; Resources, J.L.L.; Supervision, S.A.J. and R.D.F.ACKNOWLEDGMENTSThe authors would like to thank L. Benoit, N. Gurtler, K. Ceron, and E. Beaulieufor help in coding behavioral assays; Bénédict Rossi for artwork; N. Shah forbehavior analysis software; L. Becker for help with fish maintenance; N.Shah, K. Maruska, B. Grone, and C. Yang for critical reading of the manuscript;and the R.D.F. lab for useful discussions. This work was supported by NIHF32HD071755 to S.A.J.; NIH NS034950, NIH MH101373, and NSF IOS0923588 to R.D.F.; and NIH DK090065 and MH099647 to P.M.Received: December 23, 2015Revised: January 22, 2016Accepted: January 27, 2016Published: March 17, 2016REFERENCES1. Beach, F.A. (1976). Sexual attractivity, proceptivity, and receptivity infemale mammals. Horm. Behav. 7, 105–138.2. McCarthy, M.M., Bare, J.E., and vom Saal, F.S. (1986). Infanticide andparental behavior in wild female house mice: effects of ovariectomy, adrenalectomy and administration of oxytocin and prostaglandin F2 alpha.Physiol. Behav. 36, 17–23.3. Pedersen, C.A., Ascher, J.A., Monroe, Y.L., and Prange, A.J., Jr. (1982).Oxytocin induces maternal behavior in virgin female rats. Science 216,648–650.4. Rodriguez-Sierra, J.F., and Komisaruk, B.R. (1977). 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in a cichlid fish shows that dissection of gene func-tion can reveal basic control mechanisms for behav-iors in this large family of species with diverse and fascinating social systems [16, 17]. RESULTS We
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10 tips och tricks för att lyckas med ert sap-projekt 20 SAPSANYTT 2/2015 De flesta projektledare känner säkert till Cobb’s paradox. Martin Cobb verkade som CIO för sekretariatet för Treasury Board of Canada 1995 då han ställde frågan
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