ARTICLE

https://doi.org/10.1038/s41467-019-12212-7 OPEN Neurogenomic insights into paternal care and its relation to territorial aggression

Syed Abbas Bukhari1,2,3, Michael C. Saul1,6, Noelle James 4, Miles K. Bensky5, Laura R. Stein3,7, Rebecca Trapp3,8 & Alison M. Bell1,3,4,5

Motherhood is characterized by dramatic changes in brain and behavior, but less is known about fatherhood. Here we report that male sticklebacks—a small fish in which fathers

1234567890():,; provide care—experience dramatic changes in neurogenomic state as they become fathers. Some genes are unique to different stages of paternal care, some genes are shared across stages, and some genes are added to the previously acquired neurogenomic state. Com- parative genomic analysis suggests that some of these neurogenomic dynamics resemble changes associated with pregnancy and reproduction in mammalian mothers. Moreover, gene regulatory analysis identifies transcription factors that are regulated in opposite directions in response to a territorial challenge versus during paternal care. Altogether these results show that some of the molecular mechanisms of parental care might be deeply conserved and might not be sex-specific, and suggest that tradeoffs between opposing social behaviors are managed at the gene regulatory level.

1 Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana Champaign, 1206 Gregory Drive, Urbana, IL 61801, USA. 2 Illinois Informatics Institute, University of Illinois, Urbana Champaign, 616 E. Green St., Urbana, IL 61820, USA. 3 Department of Evolution, Ecology and Behavior, University of Illinois, Urbana Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, USA. 4 Neuroscience Program, University of Illinois, Urbana Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, USA. 5 Program in Ecology, Evolution and Conservation Biology, University of Illinois, Urbana Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, USA. 6Present address: Jackson Labs, 600 Main St., Bar Harbor, ME 04609, USA. 7Present address: Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Room 314, Norman, OK 73019, USA. 8Present address: Department of Biological Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907, USA. Correspondence and requests for materials should be addressed to A.M.B. (email: [email protected])

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n many species, parents provide care for their offspring, which Moreover, the basic building blocks of parental care are ancient Ican improve offspring survival. There is fascinating diversity and deeply conserved in vertebrates13. For example, the hormone in the ways in which parents care for their offspring, from prolactin was named for its essential role in lactation in mam- infant carrying behavior in titi monkeys, poison dart frogs and mals, but had functions related to parental care in fishes long spiders to provisioning of offspring in burying beetles and before mammals evolved14. Growing evidence for deep homology birds1,2. The burden of parental care does not always land of brain circuits related to social behavior15–18 suggests that the exclusively on females; in some species both parents provide care diversity of parental care among vertebrates is underlain by and in others males are solely responsible for care. changes in functionally conserved genes operating within similar Our understanding of the molecular and neuroendocrine basis neural circuits19. of parental care has been largely influenced by studies in mam- In this study, we track the neurogenomic dynamics of the mals, where maternal care is the norm. In mammals, females transition to fatherhood in male stickleback fish by measuring experience cycles of estrus, pregnancy, child birth and lactation as gene expression (RNA-Seq) in two brain regions containing they become mothers, all of which are coordinated by hormones. nodes within the social behavior network, diencephalon and tel- While maternal care is often primed by hormonal and physio- encephalon. Gene expression in experimental males is compared logical changes related to embryonic or fetal development, the across five different stages (nest, eggs and three time points after primers for paternal behavior are likely to be more subtle, such as hatching) and relative to a control group. In this species, fathers the presence of eggs or offspring3,4. Despite this subtlety, there is are solely responsible for the care of the developing offspring, and growing evidence that males can also experience changes in male sticklebacks go through a predictable series of stages as they physiology and behavior as they become fathers, some of which become fathers, from territory establishment and nest building to resemble changes in mothers5. For example, men experience mating, caring for eggs, hatching and caring for fry20. increased oxytocin6 and a drop in testosterone7 following the In addition to providing care, parents must be vigilant to birth of a child. Indeed, a recent study in burying beetles showed defend their vulnerable dependents from potential predators or that the neurogenomic state of fathers when they are the sole other threats. Tradeoffs between parental care and territory providers of care closely resembles the neurogenomic state of defense have been particularly well studied in the ecological lit- mothers8. erature, e.g.21, and parental care and territorial aggression There is taxonomic diversity in the specific behavioral mani- represent the extremes on a continuum of social behavior—from festations of care, but all care-giving parents go through a pre- strongly affiliative to strongly aversive. Therefore, an additional dictable series of stages as they become mothers or fathers, from goal of this study is to compare and contrast the neurogenomics preparatory stages prior to fertilization (e.g. territory establish- of paternal care with the neurogenomic response to a territorial ment and nest building) to the care of developing embryos (e.g. challenge. As parental care and territorial aggression are social pregnancy, incubation), to care of free-living offspring (e.g. pro- behaviors and both utilize circuitary within the social behavior visioning of nestlings, lactation, etc). Each stage is characterized network in the brain15–17, we expect to observe similarities by a set of behaviors and events, and the transition to the next between parental care and a territorial challenge at the molecular stage depends on the successful completion of the preceding stage level. However given their position at opposite ends of the con- e.g. ref. 9. The temporal ordering of stages, combined with our tinuum of social behavior, along with neuroendocrine tradeoffs understanding of the neuroendocrine dynamics of reproduc- between them22, here we test the hypothesis that opposition tion10, prompts at least three non-mutually exclusive hypotheses between parental care and territorial aggression is reflected at the about how we might expect gene expression in the brain to molecular and/or gene regulatory level. change over the course of parental care. First, because each stage Altogether results suggest that some of the molecular is characterized by a particular set of behaviors, each stage might mechanisms of parental care are deeply conserved and are not have a unique neurogenomic state associated with it (the unique sex-specific, and suggest that tradeoffs between opposing social hypothesis). Second, some of the demands of parenting remain behaviors are managed at the gene regulatory level. constant across stages, e.g. defending a nest site, therefore we might expect to see the signal of a preceding stage to persist into subsequent stages (the carryover hypothesis), resulting in shared Results genes among stages, especially between stages close together in Neurogenomic dynamics of paternal care. There were dramatic the series. Finally, extending the reasoning further, and con- neurogenomic differences associated with paternal care. A large sidering that parents must pass through one stage before pro- number of genes—almost 10% of the transcriptome—were dif- ceeding to the next, genes associated with one stage might be ferentially expressed between the control and experimental added to the previous stage as a parent proceeds through the groups over the course of the parenting period (Fig. 1a, Supple- stages (the additivity hypothesis, an extension of the carryover mentary Data 1). Within each stage, a comparable number of hypothesis).Whether changes that occur at the neurogenomic genes were up- and down-regulated. There were significant gene level can be mapped on to behaviorally defined (as opposed to expression differences between the control and experimental endogenously defined) stages of parental care is unknown. groups within both brain regions; relatively more genes were Moreover, we know little about whether there are genes that differentially expressed in diencephalon. conform to a unique, carryover or additive pattern across stages Functional enrichment analysis of the differentially expressed of care. These hypotheses provide a novel conceptual framework genes (DEGs) suggests that paternal care requires changes in for improving our understanding of parental care at the mole- energy metabolism in the brain along with modifications of cular level, and could serve as a model for studying other life immune system and transcription. Genes associated with the events that comprise a series of behaviorally defined stages, e.g. immune response were down-regulated in both brain regions and stages of territory establishment, stages of pair-bonding, stages of during most stages relative to the control group. Genes associated dispersal, etc. with energy metabolism and the adaptive component of the Unlike mammals, paternal care is relatively common in fishes: immune response were upregulated in telencephalon. Genes of the fishes that display parental care, 80% of them provide some associated with the stress response were downregulated in both form of male care, therefore fish are good subjects for under- brain regions around the day of hatching. Genes associated with standing the molecular orchestrators of paternal care11,12. energy metabolism were downregulated as the fry emerged

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a Diencephalon Telencephalon c Diencephalon Telencephalon 5 galn

4

200 3

2

1 0 3

Genes galr1

2 −200

1 Up Down Nest Eggs Early Mid Late Nest Eggs Early Mid Late 3 pgr b Diencephalon Telencephalon 2

UpDown Up Down 1

Energy Immune Adaptive Immune immune 0 metabolism response response Expression (log CPM) Nest response 6 esr1 Energy Immune Immune 5 Eggs metabolism response response 4

Immune 3 Stress response, Immune Transport Early response energy response 2 metabolism 10.0 oxt Cell Adaptive Immune Biosynthesis communication immune response,energy 7.5 Mid & gene & regulation response metabolism, expression of metabolism stress response 5.0 Immune response, Adaptive 2.5 metabolism, Late immune Metabolism cellular transport, 0.0 response Exp Con ion homeostasis Nest Eggs Early Mid Late Nest Eggs Early Mid Late

Fig. 1 Neurogenomic dynamics of paternal care. a The number of up- and down-regulated differentially expressed genes (DEGs) at each stage of paternal care in diencephalon and telencephalon. b Summary of GO-terms that were enriched in up- and down-regulated genes at each stage in the two brain regions. c The expression profile of candidate genes related to maternal care (galanin, galanin receptor 1, progesterone, estrogen receptor 1, oxytocin) across stages, with expression in the two brain regions plotted relative to the appropriate circadian control group; data points represent individual samples with means and s.e.m. indicated. Statistical significance of these genes was assessed as a pairwise contrast between a stage and its control (see Supplementary Data 1 for full list of genes; source data are in GEO GSE134508) using negative binomial distribution with generalized linear models in edgeR. Boxes surround means that are statistically different between the control and experimental condition within the stage.

(Fig. 1b, Supplementary Data 2). The expression profile of cutoff for differential expression in another stage (false negatives), particular candidate genes related to parental care are in Fig. 1c, we followed an empirical approach (as in23). We kept the cutoff with statistically significant differences between the control and for DEGs at the focal stage at FDR < 0.01 and relaxed the FDR experimental condition within a stage indicated. Altogether these threshold on the other stages (Supplementary Fig. 1). This patterns suggest that paternal care involves significant neuroge- procedure was repeated for each stage and in each brain region nomic shifts in stickleback males. separately. This analysis produced—with high statistical con- fidence—lists of DEGs that are unique to each stage (Fig. 2a), consistent with the “unique” hypothesis. Change and stability of neurogenomic state across stages.We Next, we assessed the extent to which genes were shared among used these data to assess evidence for three non-mutually exclu- different stages of paternal care by testing whether the number of sive hypotheses about how neurogenomic state might change overlapping DEGs between stages was greater than expected across stages of parental care. According to the unique hypoth- using a hypergeometric test. Consistent with the carryover esis, there is a strong effect of stage on brain gene expression and hypothesis, within each brain region, the number of overlapping little to no overlap among the genes associated with different DEGs between stages was statistically much greater than expected stages. To evaluate this hypothesis we tested whether there were by chance (Supplementary Data 3), and stages that are close DEGs that were unique to each stage, i.e. not shared with other together in the series shared more DEGs compared to stages that stages. We generated lists of genes that were differentially are further apart (Fig. 2b, Supplementary Fig. 2). expressed between the control and experimental group at each These results suggest that there are genes whose signal persists stage within each brain region. Then, we excluded the DEGs that across stages of care. We then evaluated the possibility that each were shared between stages in order to identify unique genes to new stage triggers a neurogenomic response which persists into each stage. To increase confidence that the unique genes are truly subsequent stages, i.e. that genes associated with one stage are unique to each stage, i.e. that they didn’t just barely passed the added to the previous stage as a parent proceeds through the

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a b Unique - Diencephalon Diencephalon Telencephalon Nest Eggs Early Mid Late Late Nest

25 Mid

Early

Eggs Nest 30 Eggs Eggs Early 9 Early Nest Mid

Nest Eggs Early Mid Nest Eggs Early Mid Mid

c Added shared - Diencephalon Added shared - Telencephalon Nest Eggs Early Mid Late Nest Eggs Early Mid Late Nest Nest 123 177 Eggs 3 23 Late 8 Early Unique - Telencephalon

Log (fold change) Nest 21 –2 –1 0 1 2 2 Eggs 235 8 Early Eggs 58 Mid Early 3 26 Mid Late 32 60 Fig. 2 Change and stability of neurogenomic state across stages of parental care. a There were DEGs that were only differentially expressed during one stage. Shown is a heat map depiction of the expression profile of the genes that were “unique” to each stage, showing how they were regulated in other stages, separated by stage and by brain region. b The statistical significance of the pair-wise overlap between stages within each brain region. The size of the circle is proportional to the significance of the p-value (hypergeometric test FDR) of the overlap, such that large circles indicate smaller p-values. Note that the stages closest to the focal stage tended to share more DEGs compared to stages further apart in the series. c DEGs that were added to a stage and were also differentially expressed in subsequent stages. Shown is a heat map depiction of the added shared genes for each stage, separated by brain region, showing how they were regulated across stages. Red = upregulated, blue = downregulated. Numbers on the heat maps indicate the number of genes in each heat map. Source data are in GEO GSE134508 stages. According to this hypothesis, when a parent is caring for overlap with subsequent stages, therefore except for the “nest eggs in their nest, for example, the “egg” genes are added to the added shared genes”, each of the added shared genes from the previously activated “nest” genes, and so on, in an additive previous stage(s) were subtracted from the focal stage’s added fashion. To examine this statistically, for each stage, we identified shared genes (Supplementary Fig. 3). This process generated four genes that: (1) were differentially expressed during the stage of sets of added shared genes: genes that were differentially interest; (2) were not differentially expressed during any of the expressed during the nest stage and were also differentially preceding stages; (3) were also differently expressed in a expressed during at least one subsequent stage (“nest added subsequent stage, hereafter referred to as "added shared genes". shared genes”), genes that were differentially expressed during the Only genes added during a new stage were used to test for their egg stage and were also differentially expressed during at least one

4 NATURE COMMUNICATIONS | (2019) 10:4437 | https://doi.org/10.1038/s41467-019-12212-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12212-7 ARTICLE subsequent stage but not during the nest stage (“egg added shared experience across stages of maternal care, we leveraged a recent genes”), and so on. dataset where brain gene expression was compared across a series This analysis revealed genes that became differentially of pregnancy and post-partum stages in mice (Supplementary expressed as males proceeded through different stages of paternal Data 5)31. Similar to stickleback fathers, there were both unique care and ROAST24 analysis found that the added shared genes and added shared DEGs across different stages of pregnancy and remained differentially expressed in subsequent stages in a postpartpum in mouse mothers. We then tested if the enduring statistically significant manner (Supplementary Data 4). This (added shared genes) and transient (unique) changes in suggests, for example, that there was a transcriptional signal of neurogenomic state that were experienced in stickleback fathers eggs which persisted after the egg stage. To see if the genes that were similar to the enduring and transient signals of pregnancy were added and which persisted over time were similarly and the postpartum period in mouse mothers. Specifically, we regulated across subsequent stages of paternal care, we examined compared mouse and stickleback added shared genes within the the expression profiles of the added shared genes at each stage appropriate orthogroup (Supplementary Data 6). For example, we and tested if the direction of regulation was consistent across compared 356 stickleback added shared genes within 90 stages. This analysis revealed that added shared genes were indeed orthogroups in diencephalon and 838 mouse added shared genes similarly regulated across stages (Supplementary Data 4, Fig. 2c). within 265 orthogroups in hypothalamus and found that they For example, added shared genes that were upregulated in males shared 14 orthogroups. In order to test whether those 14 shared with nests were also upregulated during subsequent stages, orthogroups is greater than expected due to chance, we employed especially during stages close to the nesting stage. To investigate a Monte Carlo based permutation approach. We did not use a this further, we calculated the probability that all genes within a regular hypergeometric test or regular permutation test here (at set of added shared genes were expressed in the same direction the orthogroup level) because each orthogroup contains more due to chance, i.e. either consistently up- or down-regulated. than one gene in both the stickleback and mouse genomes, and Then, we counted the number of genes within each set of added some of those genes were differentially expressed and others were shared genes that were concordantly expressed. We found that not. Instead, we sampled the gene sets (e.g. 356 and the number of concordantly expressed genes was greater than 838 genes in diencephalon/hypothalamus) repeatedly (105) and expected by chance (diencephalon χ2 = 1859, P < 1e-6, telence- with replacement from both species’ universes and counted the phalon χ2 = 146, df = 2, P < 1e-4). For example, 172 of the 235 overlaps at the orthogroup level. This overlap was then tested genes in the nest added shared genes in diencephalon were against the observed overlap to compute p-values, which are concordantly expressed across stages, much higher than the highly significant (Fig. 3, note that the overlap never reaches 14 expected 15 genes due to chance. The concordant expression orthogroups). Added shared genes in stickleback and mouse pattern across stages suggests that an added shared gene serves a include BDNF (a candidate gene related to anxiety, stress and similar function in different stages. depression32) and a regulator of G protein receptors RGS3 (related to insulin metabolism33). We followed the same procedure for the unique genes and did not find any evidence Pathways are not sex-specific and are deeply conserved. Some of of sharing between the two species. For example, there were 33 the candidate genes associated with female pregnancy and unique genes in four orthogroups in mouse hypothalamus and maternal care were differentially expressed in different stages of 244 unique genes in 54 orthogroups stickleback diencephalon paternal care in sticklebacks (Fig. 1c). For example, in mammals, with no overlap between them (Supplementary Data 6). levels of progesterone, estrogen and their receptors increase Altogether, the differential expression of candidate genes during pregnancy and then subside after childbirth. A similar related to maternal care along with the deep homology of the pattern was observed in the diencephalon of male sticklebacks: enduring signal of care across stages (added shared genes) suggest both estrogen receptor (esr1) and progesterone receptor (pgr) that some of the neurogenomic shifts that occur during paternal were upregulated during early hatching and then subsided care in a fish are deeply conserved and are not sex-specific. (Fig. 1c). Oxytocin (and its teleost homolog isotocin) plays an important role in social affiliation and parental care in mammals6 and fish19,25–28. Oxytocin (oxt) was upregulated in diencephalon Parenting and aggression tradeoffs at the molecular level.To when male sticklebacks were caring for eggs in their nests, and better understand how different social demands are resolved in the upregulated in telencephalon mid-way through the hatching brain, we compared these data to a previous study on the neuro- process (Fig. 1c). genomic response to a territorial challenge in male sticklebacks34, Genes that have been implicated in infanticide during parental which measured brain gene expression 30, 60 or 120 min after a 5 care in mammals were also differentially expressed in stickle- min territorial challenge. The two experiments studied behaviors at backs, where egg cannibalism is common. Galanin—a gene the opposite ends of a continuum of social behavior: paternal care implicated with infanticidal behavior in mice29—was highly provokes affiliative behavior while a territorial challenge provokes expressed in diencephalon (which includes the preoptic area) aggressive behavior, and the challenge hypothesis originally posited during the nest, eggs and early hatching stages. However, the that patterns of testosterone secretion reflects tradeoffs between galanin receptor gene was downregulated during the middle to parental care and territory defense, assuming that testosterone is late hatching stages in both brain regions (Fig. 1c). Furthermore, incompatible with parental care in males22. Subsequent studies have the progesterone receptor—which mediates aggressive behavior shown that testosterone is not always inhibitory of parental care35, toward pups in mice30—gradually declined in both brain regions and that a territorial challenge activates gene regulatory pathways as hatching progressed, and its level was lowest when all the fry that do not depend on the action of testosterone36. Regardless of the were hatched (Fig. 1c). Up-regulation of galanin during the egg specific neuromodulators or hormones, a mechanistic link between stage and down-regulation of progesterone receptor during the parental care and territory defense is likely to operate through the hatching stage could reflect how male sticklebacks inhibit social behavior network in the brain because most nodes of this cannibalistic behavior while providing care. network express receptors for neuromodulators and hormones that To test if the neurogenomic changes that we observed in are involved with both parental care and aggression37. Therefore we stickleback fathers across stages, e.g. unique and added shared used these data to assess whether there is commonality at the genes, are similar to the neurogenomic changes that mothers molecular level between aggression and paternal care. For example,

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ab Nest Eggs HEarly HMid HLate PC14 PC16 PP1 PP3 PP10 Nest Eggs HEarly HMid HLate PC14 PC16 PP1 PP3 PP10 EOG091G0Y2U | stmn4l | Stmn4 EOG090B0052 | acaca | Acacb EOG090B03S5 | cpne3 (1 of 4) | Cpne2 EOG090B01VP | gria3b | Gria2 EOG090B028A | il6st | Il6st EOG091G0BDG | si:ch211−196h16.12 | Rgs3 EOG090B00HH | MYH3 | Myh7 EOG090B0COB | bdnf | Bdnf EOG090B00HH | MYH3 | Myh13 EOG090B0EU9 | cldn7a | Cldn1 EOG090B00HH | MYH3 | Myh2 EOG090B0IGX | spen | Spen EOG090B0K1Y | vamp2 | Vamp1 EOG090B0IOF | ndufa7 | Ndufa7 EOG090B0DS7 | cnih2 | Cnih3 EOG090B01AP | b4galnt3b | B4galnt3 EOG090B02QF | prkca | Prkcb EOG090B0ACR | sox19a | Sox3 EOG090B0COB | bdnf | Bdnf –Log(fdr) EOG090B0D45 | adcyap1a | Adcyap1 8 6 EOG090B00XG | sall3a | Sall3 4 EOG090B01WI | VAV3 (1 of 2) | Vav3 2

Fig. 3 DEGs associated with shared orthogroups. Color represents the significance of differential expression between the control and experimental group (p values (−log(fdr)) across the five conditions in stickleback (left) and the five conditions in mouse (right). a shows the significance of DEGs within 14 shared orthogroups between diencephalon in stickleback and hypothalamus in mouse. b shows the significance of DEGs within nine shared orthogroups between telencephalon in stickleback and hippocampus in mouse. Source data are in GEO GSE134508

a b Paternal care Territorial challenge Territorial challenge A Territorial B challenge 30 60 120 Time (minutes)

Paternal care Nest Eggs Hatch * F F U F F F U 566 177 1941 F C U F E D ~1–7 ~5 Paternal care Time (days)

c ENSGACG00000008417

ENSGACG00000010788 ENSGACG00000009215

ENSGACG00000013094

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ENSGACG00000002226

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ENSGACG00000011629

ENSGACG00000001106

ENSGACG00000009027

ENSGACG00000011918

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ENSGACG00000009792

ENSGACG00000005610

ENSGACG00000001556 ENSGACG00000013707

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ENSGACG00000002490 ENSGACG00000009569

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ENSGACG00000020423 ENSGACG00000010416 ENSGACG00000014064

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ENSGACG00000001392 ENSGACG00000000450

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ENSGACG00000009291 ENSGACG00000000702 klf12a ENSGACG00000006913 ENSGACG00000016553 ENSGACG00000017694

ENSGACG00000018563

ENSGACG00000003199 ENSGACG00000014419 ENSGACG00000002352 ENSGACG00000020202 ENSGACG00000015883 ENSGACG00000004004 ENSGACG00000005270 ENSGACG00000015121 ENSGACG00000012218 ENSGACG00000012399 ENSGACG00000002865

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ENSGACG00000001338 ENSGACG00000004346 ENSGACG00000018829

ENSGACG00000005959 ENSGACG00000018704 ENSGACG00000013846 ENSGACG00000009264

ENSGACG00000017069 ENSGACG00000008062 ENSGACG00000017836 ENSGACG00000001289

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ENSGACG00000000703 ENSGACG00000003477 ENSGACG00000013679 ENSGACG00000000718 CRX ENSGACG00000017221 ENSGACG00000019442 ENSGACG00000014622 ENSGACG00000012208 ENSGACG00000006654 ENSGACG00000002286 ENSGACG00000018033 ENSGACG00000007585 ENSGACG00000001508 ENSGACG00000016677 ENSGACG00000001900 ENSGACG00000014887 ENSGACG00000003067 ENSGACG00000012802 ENSGACG00000001664 ENSGACG00000017959 ENSGACG00000006028 ENSGACG00000003192 ENSGACG00000006773

ENSGACG00000010350 ENSGACG00000014426 ENSGACG00000016988 ENSGACG00000005558 ENSGACG00000010211 ENSGACG00000007625 ENSGACG00000005793 ENSGACG00000003839 ENSGACG00000016678 ENSGACG00000006252 ENSGACG00000011864 ENSGACG00000020776 ENSGACG00000015660 ENSGACG00000016021

ENSGACG00000015671 ENSGACG00000010287 ENSGACG00000016316 ENSGACG00000015391 ENSGACG00000017345 ENSGACG00000003017 ENSGACG00000013854 ENSGACG00000016681 ENSGACG00000000314 ENSGACG00000000412 ENSGACG00000010270 ENSGACG00000002874 ENSGACG00000016249 ENSGACG00000012956 ENSGACG00000009422 ENSGACG00000007934 ENSGACG00000011301 ENSGACG00000002787

ENSGACG00000017900 ENSGACG00000010179 ENSGACG00000002334 ENSGACG00000018546 ENSGACG00000017612 ENSGACG00000015123 ENSGACG00000006547 ENSGACG00000001981 ENSGACG00000006479 ENSGACG00000017287 ENSGACG00000015130 ENSGACG00000001998 ENSGACG00000016811 ENSGACG00000019917 ENSGACG00000020357 ENSGACG00000006779 ENSGACG00000015964 ENSGACG00000000801 ENSGACG00000018497

ENSGACG00000000615 ENSGACG00000010875 ENSGACG00000017723 ENSGACG00000013904 ENSGACG00000016516 ENSGACG00000018547 ENSGACG00000010639

ENSGACG00000003679 ENSGACG00000008338 ENSGACG00000020178 ENSGACG00000003778 ENSGACG00000020582 ENSGACG00000016445 ENSGACG00000011827 ENSGACG00000016370 ENSGACG00000018022 ENSGACG00000016420 ENSGACG00000016253 ENSGACG00000008155 ENSGACG00000010123 ENSGACG00000008358 ENSGACG00000015381 ENSGACG00000014047 ENSGACG00000002422 ENSGACG00000019148 ENSGACG00000003937 ENSGACG00000004123 ENSGACG00000020127 ENSGACG00000005455 ENSGACG00000020017 ENSGACG00000017184

ENSGACG00000006886 ENSGACG00000012744 ENSGACG00000007705

ENSGACG00000003289 ENSGACG00000004437 ENSGACG00000008646 ENSGACG00000017121 ENSGACG00000017360 ENSGACG00000012788 ENSGACG00000019075 ENSGACG00000014245 ENSGACG00000005592 ENSGACG00000008673 ENSGACG00000008957 ENSGACG00000018521 ENSGACG00000008212 ENSGACG00000003189 ENSGACG00000020840 ENSGACG00000017207 ENSGACG00000012737 ENSGACG00000009241 ENSGACG00000012301 ENSGACG00000004207 ENSGACG00000017359 ENSGACG00000003369 ENSGACG00000010707 ENSGACG00000014580 ENSGACG00000010624 ENSGACG00000001398 ENSGACG00000018939 ENSGACG00000015748 ENSGACG00000015844 ENSGACG00000016368 ENSGACG00000006481 ENSGACG00000009761 ENSGACG00000013465 ENSGACG00000006960 ENSGACG00000003412 ENSGACG00000015051

ENSGACG00000004080 ENSGACG00000006996 ENSGACG00000020743 ENSGACG00000003919 ENSGACG00000017881

ENSGACG00000019036 ENSGACG00000010518 ENSGACG00000015259 ENSGACG00000012602 ENSGACG00000020277 ENSGACG00000003833

ENSGACG00000019125 ENSGACG00000015618 ENSGACG00000011021 ENSGACG00000010229 ENSGACG00000002357 ENSGACG00000019080 ENSGACG00000002425 ENSGACG00000017483 ENSGACG00000014002 ENSGACG00000007667 ENSGACG00000019187 ENSGACG00000018139 ENSGACG00000015840 ENSGACG00000015232 ENSGACG00000017372 ENSGACG00000007481 ENSGACG00000003615 ENSGACG00000020416 ENSGACG00000016924 ENSGACG00000001539 ENSGACG00000017255 ENSGACG00000019888 ENSGACG00000010365 ENSGACG00000011348 ENSGACG00000004568 ENSGACG00000016232 ENSGACG00000014782 ENSGACG00000016904 mef2aa ENSGACG00000007885 ENSGACG00000013917 ENSGACG00000004910 ENSGACG00000009006 ENSGACG00000010912 ENSGACG00000008918 ENSGACG00000017128 ENSGACG00000009905 ENSGACG00000002622 ENSGACG00000011974 ENSGACG00000016078 ENSGACG00000002720 ENSGACG00000008787 ENSGACG00000010085 ENSGACG00000014846 ENSGACG00000001434 ENSGACG00000011743 ENSGACG00000000628 ENSGACG00000011967 ENSGACG00000005508 ENSGACG00000003888 ENSGACG00000012138 ENSGACG00000006916 ENSGACG00000003215 ENSGACG00000012219 ENSGACG00000003340 ENSGACG00000006736 ENSGACG00000001509 ENSGACG00000001883 ENSGACG00000006581 ENSGACG00000006792 ENSGACG00000005402 ENSGACG00000008077 ENSGACG00000009438 ENSGACG00000010953 ENSGACG00000014983 ENSGACG00000006272 ENSGACG00000018040 ENSGACG00000008113 ENSGACG00000012203 ENSGACG00000007835 ENSGACG00000002573 ENSGACG00000009306 ENSGACG00000017245

ENSGACG00000003505 ENSGACG00000013146 ENSGACG00000000411 ENSGACG00000019783 ENSGACG00000007821 ENSGACG00000008778 ENSGACG00000017193 ENSGACG00000007827 ENSGACG00000006101 ENSGACG00000007122 ENSGACG00000016074 ENSGACG00000019027

ENSGACG00000010356 ENSGACG00000006572 ENSGACG00000019986

ENSGACG00000003431 ENSGACG00000012933 ENSGACG00000006082 ENSGACG00000004668 ENSGACG00000009385 ENSGACG00000000898 ENSGACG00000004873 ENSGACG00000008890 ENSGACG00000001754 ENSGACG00000001291 ENSGACG00000001295 ENSGACG00000005227 ENSGACG00000019298 ENSGACG00000014381 ENSGACG00000012028 ENSGACG00000010570 ENSGACG00000014728 ENSGACG00000014621 ENSGACG00000004757 ENSGACG00000009355 ENSGACG00000007721 ENSGACG00000019448 ENSGACG00000008279 ENSGACG00000014727 ENSGACG00000011137 ENSGACG00000017458

ENSGACG00000017597 ENSGACG00000020394 ENSGACG00000008216 ENSGACG00000004570 ENSGACG00000009959 ENSGACG00000002708

ENSGACG00000014281 ENSGACG00000007991

ENSGACG00000015372 ENSGACG00000014838 ENSGACG00000015958 ENSGACG00000013182 ENSGACG00000011932

ENSGACG00000017225 ENSGACG00000013605 ENSGACG00000010970 ENSGACG00000015747 ENSGACG00000008947 ENSGACG00000001665 ENSGACG00000015386 ENSGACG00000005822 ENSGACG00000013353 ENSGACG00000009797 ENSGACG00000002228 ENSGACG00000007776 ENSGACG00000006863 ENSGACG00000008402 ENSGACG00000011084 ENSGACG00000011995 ENSGACG00000006392 ENSGACG00000015734 ENSGACG00000003513 ENSGACG00000012516 ENSGACG00000012157 ENSGACG00000014163

ENSGACG00000005058 ENSGACG00000003813 ENSGACG00000017176 ENSGACG00000008329 ENSGACG00000018910 ENSGACG00000017369 ENSGACG00000004088 ENSGACG00000019611 ENSGACG00000018854 ENSGACG00000019340 ENSGACG00000005966 ENSGACG00000008405 ENSGACG00000017885 ENSGACG00000009794 ENSGACG00000008792 ENSGACG00000004399 ENSGACG00000004914 ENSGACG00000010700 ENSGACG00000011793 ENSGACG00000009888 ENSGACG00000007443 ENSGACG00000020076 ENSGACG00000017882 ENSGACG00000013512 ENSGACG00000012436 ENSGACG00000011762 ENSGACG00000007335 ENSGACG00000018364 ENSGACG00000012867 nfixa ENSGACG00000013069 ENSGACG00000007641 ENSGACG00000008199 ENSGACG00000004154 ENSGACG00000011207 ENSGACG00000007319 ENSGACG00000015979 ENSGACG00000014720 ENSGACG00000014766 ENSGACG00000003160

ENSGACG00000015271 ENSGACG00000014579 ENSGACG00000017017 ENSGACG00000018664 ENSGACG00000019462 ENSGACG00000014202 ENSGACG00000015990

ENSGACG00000006854 ENSGACG00000017015 ENSGACG00000010385 ENSGACG00000010079 ENSGACG00000009821 ENSGACG00000015648 ENSGACG00000005950 ENSGACG00000014009 ENSGACG00000020406 ENSGACG00000013387 ENSGACG00000018709 ENSGACG00000019789 ENSGACG00000002014 ENSGACG00000007995 ENSGACG00000002778 ENSGACG00000001065 ENSGACG00000016538 ENSGACG00000007843 ENSGACG00000017305 ENSGACG00000020307 ENSGACG00000014956 ENSGACG00000009115 ENSGACG00000007968 ENSGACG00000002995 ENSGACG00000007617 ENSGACG00000018855 ENSGACG00000013900 ENSGACG00000004655 ENSGACG00000016661 ENSGACG00000014290 ENSGACG00000002824 ENSGACG00000008491

ENSGACG00000014234 ENSGACG00000006049 ENSGACG00000009373 ENSGACG00000007978 ENSGACG00000016858 ENSGACG00000010820 ENSGACG00000014616 ENSGACG00000007197 ENSGACG00000014591 ENSGACG00000009671 ENSGACG00000008541 ENSGACG00000008752 ENSGACG00000013764 ENSGACG00000014859 ENSGACG00000020890 ENSGACG00000016713 ENSGACG00000010864 ENSGACG00000007196 ENSGACG00000003033 ENSGACG00000020297 ENSGACG00000002430 ENSGACG00000000498 ENSGACG00000020171 ENSGACG00000020135 ENSGACG00000020583 ENSGACG00000005648 ENSGACG00000010481

ENSGACG00000008647 ENSGACG00000013667 ENSGACG00000011225 ENSGACG00000004222 ENSGACG00000016413 ENSGACG00000020694 ENSGACG00000010574

ENSGACG00000014632 ENSGACG00000010857 ENSGACG00000017972 ENSGACG00000013357 ENSGACG00000008337 ENSGACG00000018827 ENSGACG00000011118 ENSGACG00000010185 ENSGACG00000020896 ENSGACG00000011250 ENSGACG00000017136 ENSGACG00000008773 ENSGACG00000017463 ENSGACG00000020332 ENSGACG00000004750 ENSGACG00000017098 ENSGACG00000019417 ENSGACG00000003588 ENSGACG00000017213 ENSGACG00000006189 ENSGACG00000020131 ENSGACG00000003274 ENSGACG00000004227 ENSGACG00000008363 ENSGACG00000020913 ENSGACG00000001975 ENSGACG00000005880 ENSGACG00000013218 ENSGACG00000018145

ENSGACG00000003653 ENSGACG00000009096 ENSGACG00000013875 ENSGACG00000017243 ENSGACG00000011239 ENSGACG00000002152 neurod1 ENSGACG00000016915 ENSGACG00000005612 ENSGACG00000004931 ENSGACG00000009112 ENSGACG00000010683

ENSGACG00000014124 KLF7 ENSGACG00000008204 ENSGACG00000019096 ENSGACG00000008076 ENSGACG00000007358 ENSGACG00000012897 ENSGACG00000006337 ENSGACG00000002124 ENSGACG00000007829 ENSGACG00000010752 ENSGACG00000010111

ENSGACG00000009831 ENSGACG00000012026 ENSGACG00000008601 ENSGACG00000018373 ENSGACG00000010804 ENSGACG00000011942 ENSGACG00000004580

ENSGACG00000002064 ENSGACG00000009909 ENSGACG00000002889 ENSGACG00000003992 ENSGACG00000011206 ENSGACG00000009189 ENSGACG00000005015 ENSGACG00000005849 ENSGACG00000008378 ENSGACG00000016241 ENSGACG00000000319

ENSGACG00000005980 ENSGACG00000008542 ENSGACG00000020725 ENSGACG00000007959 ENSGACG00000012091 ENSGACG00000011189 ENSGACG00000000599 ENSGACG00000017441 ENSGACG00000001411 ENSGACG00000016682 ENSGACG00000001712 ENSGACG00000012340 ENSGACG00000006882 ENSGACG00000007006 ENSGACG00000006322 ENSGACG00000009461 ENSGACG00000006780 ENSGACG00000020589 ENSGACG00000003824 ENSGACG00000011998

ENSGACG00000019990 ENSGACG00000000028 ENSGACG00000004084 ENSGACG00000001161 ENSGACG00000018672 ENSGACG00000002928 ENSGACG00000019529 ENSGACG00000009256

ENSGACG00000015987 ENSGACG00000000383

ENSGACG00000012606 ENSGACG00000007333 ENSGACG00000020281 ENSGACG00000017820 ENSGACG00000008956 ENSGACG00000002672 ENSGACG00000014991 ENSGACG00000013470 ENSGACG00000003678 ENSGACG00000002248 ENSGACG00000015393 ENSGACG00000016537 ENSGACG00000019206 ENSGACG00000016365 ENSGACG00000001429 ENSGACG00000014208 NR3C1 ENSGACG00000010299 ENSGACG00000006600 ENSGACG00000014194

ENSGACG00000016266 ENSGACG00000010158 ENSGACG00000009540 ENSGACG00000001673 ENSGACG00000016342 ENSGACG00000017070 ENSGACG00000015525

ENSGACG00000013298 ENSGACG00000018034 ENSGACG00000018943 ENSGACG00000009227 ENSGACG00000010684 ENSGACG00000013333

ENSGACG00000013555 ENSGACG00000002854

ENSGACG00000007543 ENSGACG00000016640 ENSGACG00000009957

ENSGACG00000011685 ENSGACG00000011555 ENSGACG00000005947 ENSGACG00000013159

ENSGACG00000003623 ENSGACG00000010801

ENSGACG00000000374

ENSGACG00000015177 ENSGACG00000010878

ENSGACG00000000108

ENSGACG00000002432 ENSGACG00000004470

ENSGACG00000018603

ENSGACG00000004072 ENSGACG00000008795 ENSGACG00000020684 ENSGACG00000020876 ENSGACG00000001336 ENSGACG00000013522 ENSGACG00000019834 ENSGACG00000013279 ENSGACG00000000664 ENSGACG00000020774 ENSGACG00000012870 ENSGACG00000005951 ENSGACG00000020128 ENSGACG00000018487 ENSGACG00000011215 ENSGACG00000010998 ENSGACG00000006632 ENSGACG00000004966 ENSGACG00000000143 ENSGACG00000008451 ENSGACG00000001703 ENSGACG00000009991

ENSGACG00000013981

ENSGACG00000018450

ENSGACG00000001892 ENSGACG00000016580

ENSGACG00000006074

ENSGACG00000009608

ENSGACG00000000364 ENSGACG00000007252 ENSGACG00000005834 ENSGACG00000008175

ENSGACG00000016626 ENSGACG00000003520 ENSGACG00000011598 ENSGACG00000005347

ENSGACG00000012890 ENSGACG00000007760 ENSGACG00000019476 ENSGACG00000017920

ENSGACG00000011378

ENSGACG00000006370

ENSGACG00000007982 ENSGACG00000019282 ENSGACG00000011940 ENSGACG00000016483 ENSGACG00000018248

ENSGACG00000000749 ENSGACG00000014360 ENSGACG00000006037

ENSGACG00000017526

ENSGACG00000015284

ENSGACG00000010708 ENSGACG00000008391 ENSGACG00000011135 ENSGACG00000020313

ENSGACG00000010371

ENSGACG00000016070

ENSGACG00000002870

ENSGACG00000016943

ENSGACG00000018292

ENSGACG00000018392

ENSGACG00000014236 ENSGACG00000015635

ENSGACG00000010594 ENSGACG00000015312

ENSGACG00000002760 ENSGACG00000020458 ENSGACG00000013373 ENSGACG00000008464 ENSGACG00000012267

ENSGACG00000005416

ENSGACG00000006999 ENSGACG00000009548

ENSGACG00000008413 ENSGACG00000016527

ENSGACG00000020111 ENSGACG00000005573

ENSGACG00000012060 ENSGACG00000002418 ENSGACG00000015589 ENSGACG00000012216 ENSGACG00000018896 ENSGACG00000011863 ENSGACG00000012885 ENSGACG00000000510

ENSGACG00000015378 ENSGACG00000016894

ENSGACG00000015011

ENSGACG00000018938

ENSGACG00000016314

ENSGACG00000018294 ENSGACG00000004484

ENSGACG00000011384

ENSGACG00000002843 ENSGACG00000002626

ENSGACG00000020693 ENSGACG00000015573

ENSGACG00000018632 ENSGACG00000008433 ENSGACG00000010140

ENSGACG00000003338

ENSGACG00000006229 ENSGACG00000003844

ENSGACG00000019956 ENSGACG00000013251

ENSGACG00000009846 ENSGACG00000015779 ENSGACG00000018986

ENSGACG00000004771 ENSGACG00000020575 ENSGACG00000020511 ENSGACG00000017398

ENSGACG00000016255 ENSGACG00000011185 ENSGACG00000011556 ENSGACG00000003082 ENSGACG00000011802 ENSGACG00000006618

ENSGACG00000007295 ENSGACG00000006986

ENSGACG00000018323 ENSGACG00000016300 ENSGACG00000008116 ENSGACG00000015349 ENSGACG00000011926

ENSGACG00000018833 ENSGACG00000005571

ENSGACG00000014345

ENSGACG00000002028 ENSGACG00000019546 ENSGACG00000008964 ENSGACG00000002585 ENSGACG00000002524

ENSGACG00000017682 ENSGACG00000000538 ENSGACG00000018865 ENSGACG00000009051 ENSGACG00000000096

ENSGACG00000013947 ENSGACG00000006756 ENSGACG00000020321 ENSGACG00000016642

ENSGACG00000007226

ENSGACG00000013013 ENSGACG00000003118

ENSGACG00000000506 ENSGACG00000017695

ENSGACG00000007687 ENSGACG00000001600 ENSGACG00000006867 ENSGACG00000013137

ENSGACG00000010155

ENSGACG00000005679

ENSGACG00000004799 ENSGACG00000009575

ENSGACG00000010672

ENSGACG00000000367 ENSGACG00000015995

ENSGACG00000012230

ENSGACG00000013822 ENSGACG00000012384

ENSGACG00000016017 ENSGACG00000001862 ENSGACG00000004885

ENSGACG00000004685

ENSGACG00000015401

ENSGACG00000007999 ENSGACG00000005755 ENSGACG00000009899

ENSGACG00000015813 ENSGACG00000012041 ENSGACG00000013839 ENSGACG00000018740 ENSGACG00000010554 ENSGACG00000007535 ENSGACG00000003220 ENSGACG00000014714 ENSGACG00000003964

ENSGACG00000007386 ENSGACG00000008410 ENSGACG00000009632 ENSGACG00000011299

ENSGACG00000016214 ENSGACG00000007542

ENSGACG00000007272 ENSGACG00000013708 ENSGACG00000005112 ENSGACG00000007627 ENSGACG00000002910

ENSGACG00000016512

ENSGACG00000007913 ENSGACG00000003036 ENSGACG00000008549 ENSGACG00000013411

ENSGACG00000007305 ENSGACG00000017699 ENSGACG00000016275 ENSGACG00000020430 Paternal care ENSGACG00000004341 ENSGACG00000013231 ENSGACG00000009494 ENSGACG00000016482

ENSGACG00000016862 ENSGACG00000013922 ENSGACG00000020195 ENSGACG00000001987 ENSGACG00000000061 ENSGACG00000011398

ENSGACG00000016444

ENSGACG00000006279 ENSGACG00000003344 ENSGACG00000013501 ENSGACG00000003436

ENSGACG00000015373 ENSGACG00000012650 ENSGACG00000012628

ENSGACG00000004954

ENSGACG00000008982

ENSGACG00000004496 ENSGACG00000020188 ENSGACG00000019222 ENSGACG00000008733 ENSGACG00000019842 ENSGACG00000008073 ENSGACG00000007857 ENSGACG00000012350 ENSGACG00000000999 ENSGACG00000005222

ENSGACG00000004306 ENSGACG00000015432

ENSGACG00000015157

ENSGACG00000008851

ENSGACG00000001148

ENSGACG00000007713

ENSGACG00000001483

ENSGACG00000006527 ENSGACG00000008751 ENSGACG00000006532

ENSGACG00000016565 ENSGACG00000018911 ENSGACG00000006748

ENSGACG00000003466 ENSGACG00000020311

ENSGACG00000007204 ENSGACG00000006914

ENSGACG00000003912

ENSGACG00000008483 ENSGACG00000020545

ENSGACG00000004387

ENSGACG00000020361 ENSGACG00000007420 ENSGACG00000003106 ENSGACG00000020762 ENSGACG00000007398

ENSGACG00000019590 ENSGACG00000004302 ENSGACG00000007802 Territorial challenge ENSGACG00000004267 ENSGACG00000003869

ENSGACG00000005480

ENSGACG00000016046

ENSGACG00000008573 ENSGACG00000009390 ENSGACG00000013419 ENSGACG00000004889 ENSGACG00000020734 ENSGACG00000007086

ENSGACG00000020030

ENSGACG00000000419

ENSGACG00000008896

ENSGACG00000004839 ENSGACG00000002828

ENSGACG00000016854

ENSGACG00000010920 ENSGACG00000007441

ENSGACG00000002299 ENSGACG00000009468 ENSGACG00000011447

pou1f1 ENSGACG00000014618

ENSGACG00000010769

ENSGACG00000017501

ENSGACG00000000613 ENSGACG00000005059

ENSGACG00000013195

ENSGACG00000008195 ENSGACG00000014295 ENSGACG00000002897 Both ENSGACG00000001525 ENSGACG00000010292

ENSGACG00000006709

ENSGACG00000011152

ENSGACG00000012671 ENSGACG00000010133 ENSGACG00000009153 ENSGACG00000019718

ENSGACG00000020175

ENSGACG00000013224

ENSGACG00000016592 ENSGACG00000018317

ENSGACG00000001173

LHX3 ENSGACG00000000803

ENSGACG00000001526

ENSGACG00000004422

ENSGACG00000001968 nr5a1b ENSGACG00000007914

Fig. 4 The regulatory dynamics of territorial challenge and paternal care. a Experimental time course sampling design in the two experiments. b Overlap between territorial aggression and paternal care DEGs. DEGs were pooled across time points and brain regions. c ASTRIX-generated transcriptional regulatory network. Each node represents a transcription factor or a predicted transcription factor target gene. Oversized nodes are transcription factors where the size of the node is proportional to the number of targets. Transcription factors whose targets are significantly enriched in either or both experiments are highlighted with different colors. Stickleback imaged drawn by MB. Source data are in GEO GSE134508 shared genes could reflect general processes such as the response to the animal is responding to a positive (parental care) versus nega- a social stimulus, while genes that are specifictoanexperiment tive (territorial challenge) social stimulus. could reflect the unique biology of paternal care versus territorial To compare the neurogenomics of paternal care and the aggression. Alternatively, there might be a set of genes that is response to a territorial challenge at the gene level, we pooled associated with both parental care and territorial aggression, but genes that were differentially expressed in the experimental those genes are regulated in different ways depending on whether compared to the control group (FDR < 0.01) across time points,

6 NATURE COMMUNICATIONS | (2019) 10:4437 | https://doi.org/10.1038/s41467-019-12212-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12212-7 ARTICLE

Territorial Paternal care challenge D30 D60 D120 T30 T60 T120 Dnest Deggs Dearly Dmid Dlate Tnest Teggs Tearly Tmid Tlate klf7b Territorial Paternal care challenge nfixa pgbd5 rly dlgap1a vat1 Dnest Deggs Dea Dmid Dlate Tnest Teggs Tearly Tmid Tlate D30 D60 D120 T30 T60 T120 gnao1a neurod SPTBN1 ENSGACG00000011743 chst11 MIER3 epha4b klf12b trim9 klf7b pcdh1a mef2aa celf5b NR3C1 (2 of 2) nfixa NR3C1 (2 of 2) crx nr5a1b ARHGAP44 (2 of 2) CELF6 mef2d vamp2 Key ENSGACG00000014632 0.4 mbnl3 0.2 kcnb1 0 −0.2 DLG4 (2 of 2) −0.4 nudt14

Fig. 5 Shared regulators of a territorial challenge and paternal care. The panel on the left shows the expression pattern of the 10 transcription factors that were enriched in both experiments (Fig. 4). Columns are conditions within the two experiments (30, 60 or 120 min after a territorial challenge, the five stages of paternal care in diencephalon (D) or telencephalon (T)). Note that 8 of the shared transcription factors were regulated in opposite directions and in different brain regions in the two experiments. The two panels on the right show the expression pattern of two examples of shared, differentially regulated transcription factors (Klf7b and NR3C1) and their targets across all of the conditions. Source data are in GEO GSE134508 stages and brain regions within each experiment, which resulted telencephalon in response to a territorial challenge and down- in two sets of genes associated with either a territorial challenge or regulated in diencephalon during parental care. These findings paternal care (Fig. 4a). There were 177 genes that were shared point to the molecular mechanisms by which transcription factors between the two experiments (Fig. 4b); this overlap is highly might differentially modulate the social behavior network15–17 in statistically significant (hypergeometric test, fdr < 1e-10). the brain to manage conflicts between paternal care and territory To identify genes that were unique to each experiment while defense. guarding against false positives, we adopted the same empirical approach as described above (Supplementary Fig. 1). There were 153 genes unique to territorial challenge and 764 genes unique to Discussion paternal care and these unique genes were enriched with non- While maternal care has long been recognized as an intense period overlapping functional categories (Supplementary Data 7). For when the maternal brain is reorganized41,42, our results suggest that example, some of the genes that were unique to a territorial paternal care also involves significant neurogenomic shifts. Many of challenge were related to sensory perception and tissue develop- the neuroendocrine changes that are experienced by mammalian ment, whereas some of the genes that were unique to paternal mothers are driven by endogenous cues during pregnancy, birth care were related to oxidative phosphorylation and energy and lactation, and are required for fetal growth and metabolism, which might reflect the high metabolic needs of development31,43, with the neural circuits necessary for maternal males as they are providing care38. care being primed by hormones during pregnancy and the post- The large number of genes that were differentially expressed both partum periods42. Our results suggest that males can also experi- during paternal care and in response to a territorial challenge ence dramatic neuromolecular changes as they become fathers, even prompted us to test for evidence of their common regulation at the in the absence of ovulation, parturition, postpartum events and gene regulatory level. Therefore, we used the data from both lactation and their associated hormone dynamics5. We observed experiments to build a transcriptional regulatory network and asked dramatic neurogenomic changes in males in response to cues for if there are transcription factors whose targets were significantly care that are exogenous (e.g. the presence of nesting material) and associated with the DEG sets from the paternal care experiment, the social (e.g. the presence of eggs or the hatching of fry). Such dra- territorial challenge experiment or both experiments (Fig. 4, matic neurogenomic shifts associated with paternal care might be Supplementary Data 8). There were 10 transcription factors that especially likely to occur in species when fathers are the sole pro- were significantly enriched in both experiments. Eight out of 10 viders of parental care, such as in sticklebacks. The effects might not transcription factors were regulated in opposite directions in at least be as strong in biparental systems where fathers contribute less. one of the conditions in the two experiments (Fig. 5). Two of the Consistent with this hypothesis, in burying beetles, when males transcription factors that were regulated in opposite directions were the sole providers of care, their brain gene expression profile (NR3C1 and klf7b) have been implicated with social behavior in was similar to mothers, but when they were biparental, fathers’ other studies (the glucocorticoid receptor NR3C1 and psychosocial neurogenomic state was less similar to mothers’8. stress during pregnancy39; klf7b and austim spectrum disorder40). A key challenge for care-giving parents is to defend their home These patterns suggest that for some genes, different salient and vulnerable offspring from threats, such as territorial intru- experiences—providing paternal care and territorial aggression— ders. Behavioral trade-offs between parental care and territory trigger opposite gene regulatory responses. defense are well-documented35 and work in this area has been Interestingly, the transcription factors showing the opposite influenced by the challenge hypothesis22, which originally posited expression pattern were differentially expressed in different brain that androgens mediate the conflict between care and aggression. regions in the two experiments. Specifically, shared transcription By comparing the neurogenomic dynamics of paternal care and factors and their predicted targets were up-regulated in the response to a territorial challenge, our work offers insights

NATURE COMMUNICATIONS | (2019) 10:4437 | https://doi.org/10.1038/s41467-019-12212-7 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12212-7 into the gene regulatory mechanisms by which animals resolve The finding of partial commonality between paternal care in a these conflicting demands. Our results suggest that opposing fish and maternal care in a mammal adds to the growing body of social experiences acting over different time scales—providing work showing that the underlying neural and molecular paternal care over the course of weeks versus responding to a mechanisms related to parental care might have been repeatedly territorial challenge over the course of minutes to hours—trigger recruited during the evolution and diversification of parental opposite gene regulatory responses. In particular, an analysis of care19,47. Indeed, our results suggest that so-called “pregnancy the predicted gene regulatory network identified transcription hormones” and added shared genes (for instance BDNF and G factors that were significantly enriched both following paternal protein regulators, RGS3) might have been serving functions care and in response to a territorial challenge, and the majority of related to care giving long before the evolution of mammals, and the transcription factors (and their targets) were regulated in that these mechanisms operate just as well in fathers as they do in opposite directions in the two experiments (Fig. 5). mothers. These commonalities with maternal care in mammals While previous studies have explored circuit-level changes in the suggest that the neurogenomic shifts that occur during paternal social behavior network in response to different social stimuli15,our care in a fish might be deeply conserved and might not be sex- results point to the molecular basis of differential modulation of the specific. Animals have been dealing with the problem of how to social behavior network: the transcription factors showing the improve offspring survival (as well as avoiding filial cannibalism) opposite expression pattern were differentially expressed in different for a long time; our results suggest that they have relied on brain regions in the two experiments. Specifically, shared tran- ancient molecular substrates to solve it. scription factors and their predicted targets were up-regulated in telencephalon in response to a territorial challenge and down- regulated in diencephalon during parental care. These findings Methods Sticklebacks. In sticklebacks, paternal care is necessary for offspring survival and suggest the molecular mechanisms by which transcription factors is influenced by prolactin48, and the main androgen in fishes (11KT) does not 15–17 might differentially modulate the social behavior network in inhibit paternal care in this species49. Paternal care in sticklebacks is costly both in the brain to manage conflicts between paternal care and territory terms of time and energy38, infanticide and cannibalism are common20, and males defense. A similar pattern was observed at the transcriptomic must be highly vigilant to challenges from predators and rival males while caring (rather than gene regulatory) level when neurogenomic states were for their vulnerable offspring. Adult males were collected from Putah Creek, CA, a freshwater population, in compared between territorial aggression and courtship in male spring 2013, shipped to the University of Illinois where they were maintained in the threespined sticklebacks: some genes that were upregulated after a lab on a 16:8 (L:D) photoperiod and at 18 °C in separate 9-l tanks. Males were territorial challenge were downregulated after a courtship oppor- provided with nesting material including algae, sand and gravel and were visually tunity44. These results are also consistent with a detailed mechan- isolated from neighbors. In order to track transcriptional dynamics associated with becoming a father, we istic study which showed that transcription factors play a role in sampled males for brain gene expression profiling at five different points during the setting up neural circuits to mediate opposing behaviors45. reproductive cycle (n = 5 males per time point): nest, eggs, early hatching, middle Altogether our analysis of changes in neurogenomic state hatching and late hatching (control: reproductively mature males with no nests). across stages of paternal care offers support for all three Males in the nest condition had a nest but had not yet mated. Males in the eggs condition were sampled four days after their eggs were fertilized. Because males in the hypotheses proposed. For example, consistent with the unique eggs condition were sampled four days after mating, the transcriptomic effects of hypothesis, there were genes that were unique to each stage. mating are likely to have attenuated by the time males were sampled at this stage. Genes exhibiting transient, stage-specific differential expression Hatching takes place over the course of the 5th day after fertilization, and a previous might be involved in facilitating the next stage, priming and/or study found that brain activation as assessed by Egr-1 expression was highest while male sticklebacks were caring for fry as compared to males with nests or eggs50.In responding to a particular event or stimulus during that stage, e.g. order to capture males’ response to the new social stimulus of their fry (see51), we the arrival of offspring. Whether genes that were unique to a focused on three time points on the day of hatching, which capture the start of the particular stage and not differentially expressed in other stages are hatching process (9 a.m.), when approximately half of the clutch is hatched (1 p.m.) a cause of future behavior or consequence of past behavior is and when all of the eggs have hatched (5 p.m.). Males in the nest, eggs and early hatching conditions were sampled at 9 a.m., unknown. We also found support for the carryover and additivity males in the mid-hatching condition were sampled at 1 p.m. and males in the late hypotheses: elements of an acquired neurogenomic state persisted hatching condition were sampled at 5 p.m. Males in these conditions were into subsequent stages, which suggests that the events and compared to reproductively mature circadian-matched control males that did not behaviors that characterize a particular stage of paternal care (e.g. have a nest (n = 5 males per control group). Wild-caught females from the same fi finishing a nest, the arrival of eggs, hatching) trigger a neuroge- population were used as mothers. Males were quickly netted and sacri ced by decapitation within seconds. All methods were approved by the IACUC of the nomic state that persists, perhaps for as long as those events and University of Illinois at Urbana-Champaign (#15077). behaviors continue. Genes whose expression persists across stages could be involved in maintaining the previous neurogenomic state, and/or reflect the constant demands of parenthood, e.g. the RNA sequencing. Heads were flash frozen in liquid nitrogen and the telencephalon nest must be maintained across all stages of care. and diencephalon were carefully dissected and placed individually in Eppendorf tubes containing 500 μL of TRIzol Reagent (Life Technologies). Total RNA was isolated Moreover, our results suggest that changes in neurogenomic immediately using TRIzol Reagent according to the manufacturer’s recommendation state in a fathering fish might share commonalities at the mole- and subsequently purified on columns with the RNeasy kit (QIAGEN). RNA was cular level with the neurogenomic changes associated with eluted in a total volume of 30 μL in RNase-free water. Samples were treated with DNase (QIAGEN) to remove genomic DNA during the extraction procedure. RNA maternal care in a mammal. The number of orthologous genes fi 31 quantity was assessed using a Nanodrop spectrophotometer (Thermo Scienti c), and that were shared across stages of maternal care in mice and RNA quality was assessed using the Agilent Bioanalyzer 2100 (RIN 7.5–10); one paternal care in sticklebacks was greater than expected due to sample was excluded because of low RNA quality. RNA was immediately stored at chance. This suggests that the neurogenomic state that is main- −80 °C until used in sequencing library preparation. tained across pregnancy and the post partum period in mice, for The RNAseq libraries were constructed with the TruSeq® Stranded mRNA HT (Illumina) using an ePMotion 5075 robot (Eppendorf). Libraries were quantified example, at least partially resembles the neurogenomic state that on a Qubit fluorometer, using the dsDNA High Sensitivity Assay Kit (Life is maintained while a male stickleback is caring for eggs and while Technologies), and library size was assessed on a Bioanalyzer High Sensitivity DNA the eggs are hatching. These results suggest that maternal and chip (Agilent). Libraries were pooled and diluted to a final concentration of 10 nM. paternal care might share similarities at the molecular level, and Final library pools were quantified using real-time PCR, using the Illumina fi compatible kit and standards (KAPA) by the W. M. Keck Center for Comparative this nding is consistent with other studies showing that parental and Functional Genomics at the Roy J. Carver Biotechnology Center (University of males and females can use the same hormones and molecular Illinois). Single-end sequencing was performed on an Illumina HiSeq 2500 mechanisms to activate the same pathways in the brain46. instrument using a TruSeq SBS sequencing kit version 3 by the W. M. Keck Center

8 NATURE COMMUNICATIONS | (2019) 10:4437 | https://doi.org/10.1038/s41467-019-12212-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12212-7 ARTICLE for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Overlap significance. We tested the significance of unique and added shared DEGs Center (University of Illinois). The 79 libraries were sequenced on 27 lanes. between stickleback and mouse at the orthogroup level. We used Monte Carlo repeated random sampling to determine if an observed orthogroup overlap between species was statistically significant at P < 0.0561. For example, suppose tà is the 52 RNA Seq informatics. FASTQC version 0.11.3 was used to assess the quality of observed orthogroup overlap between the stickleback and the mouse gene lists and n1 the reads. RNA-seq produced an average of 60 million reads per sample (Sup- and n2 are gene set sizes respectively. We repeatedly and randomly drew samples of plementary Data 9). We aligned reads to the Gasterosteus aculeatus reference size n1 from the stickleback genome and samples of n2 from the mouse genome for M = 5 genome (the repeat masked reference genome, Ensembl release 75), using TopHat times (M 10 ) with replacement and detected an overlap ti for each iteration of M (2.0.8)53 and Bowtie (2.1.0)54. Results of the TopHat alignment were largely in and computed an estimated p-value using the following equation, 55 P agreement with results from HISAT2 (Supplementary Fig. 4). Reads were M à 1 þ ¼ ItðÞ t assigned to features according to the Ensembl release 75 gene annotation file estimate } ¼ i 1 i ð1Þ (http://ftp.ensembl.org/pub/release-75/gtf/gasterosteus_aculeatus/). We used the 1 þ M default settings in all the programs unless otherwise noted. where I(.) is an indicator function.

Transcriptional regulatory network (TRN) analysis. ASTRIX uses gene expres- Defining DEGs. HTSeq v0.6.156 read counts were generated for genes using sion data to identify regulatory interactions between transcription factors and their stickleback genome annotation. Any reads that fell in multiple genes were excluded target genes. A previous study validated ASTRIX-generated TF-target associations from the analysis. We included genes with at least 0.5 count per million (cpm) in at using data from ModENCODE, REDfly, and DROID databases62. The predicted least five samples, resulting in 17,659 and 17,463 genes in diencephalon and tele- targets of TFs were defined as those genes that share very high mutual information (P ncephalon, respectively. Count data were TMM (trimmed mean of M-values) − <10 6) with a TF, and can be predicted quantitatively with high accuracy (Root normalized in R using edgeR v3.16.557. Samples separated cleanly by brain region Mean Square Deviation (RMSD) < 0.33 i.e prediction error less than 1/3rd of each on an MDS plot; we did not detect any outliers. To assess differential expression, gene expression profile’s standard deviation. The list of putative TFs in the stickleback pairwise comparisons between experimental and control conditions were made at genome was obtained from the Animal Transcription Factor Database. Given TFs and each stage using appropriate circadian controls. Because the nest, eggs and early targets sets ASTRIX infers a genome-scale TRN model capable of making quantitative stages were all sampled at 9 a.m., their expression was compared relative to the predictions about the expression levels of genes given the expression values of the same 9 a.m. control group. transcription factors. The ASTRIX algorithm was previously used to infer a TRN Diencephalon and telencephalon were analyzed separately in edgeR v3.16.5. A – models for honeybee, mouse and sticklebacks34,62 64. ASTRIX identified transcription tagwise dispersion estimate was used after computing common and trended factors that are central actors in regulating aggression, maturation and foraging dispersions. To call differential expression between treatment groups, a “glm” behaviors in the honeybee brain62. approach was used. We adjusted actual p-values via empirical FDR, where a null Here we used ASTRIX to infer a joint gene regulatory network by combining distribution of p-values was determined by permuting sample labels for 500 times for gene expression profiles from a previous study on the transcriptomic response to a each tested contrast and a false discovery rate was estimated58. Similarities across territorial challenge in male sticklebacks34 with the data from this experiment. stages of care was assessed using hypergeometric tests and PCA (Supplementary Combining the two datasets increased statistical power to help identify modules Fig. 2). that are shared and unique to the two experiments. Transcription factors that are For a fair comparison between our study and Ray et al.31, we reanalyzed the Ray predicted to regulate DEGs in either experiment were determined according to et al., gene expression dataset by applying the same model, dispersion estimates whether they had a significant number of targets as assessed by a Bonferroni FDR- and false discovery rate procedures. corrected hypergeometric test.

Unique genes. One of the goals of this study was to identify genes that uniquely Functional analysis. We derived GO assignments, using protein family annota- characterized a particular state, e.g. to a particular stage of paternal care, or to either tions from the database PANTHER65. Stickleback protein sequences were the territorial challenge or the paternal care experiment. To address the possibility that blasted against all genomes in the database (PANTHER 9.0 85 genomes). This putative unique genes barely passed the cutoff for differential expression in another procedure assigns proteins to PANTHER families on the basis of structural state (false negatives), we adopted an empirical approach, as in ref. 23.Wekeptthe information as well as phylogenetic information. Genes were then annotated cutoff for DEGs at the focal state at FDR < 0.01 and relaxed the FDR cutoff on the using GO information derived from the 85 sequenced genomes in the PANTHER other states (see Supplementary Fig. 1 for an explanation of this procedure). This database. procedure was repeated for each state and in each brain region separately. GO analysis were performed in R using TopGo v.2.16.0 and Fisher’sexacttest.A p-value cut off of <0.01 was used to select for significantly enriched functional terms wherever possible. We summarized the GO terms into larger and general categories to Added shared genes. We wanted to know how many of the genes that were get a general overview of the underlying biology. Terms were grouped together if they differentially expressed in one stage remained differently expressed in the sub- were in a similar pathway and/or based on semantic similarity. GO enrichments along sequent stages (added shared genes). To find added shared genes, we first selected with their respective p-values are in Supplementary Data 2 and 7. those stages which had significant pairwise overlap between them (FDR < 0.05, hypergeometric test). Only those genes were tested for overlap with subsequent Reporting summary. Further information on research design is available in stages; in order to qualify as an added shared genes for a particular stage, the gene the Nature Research Reporting Summary linked to this article. could not be differentially expressed during a preceding stage and had to be dif- ferentially expressed during a subsequent stage, but not necessarily the stage immediately following that particular stage. Except for the first stage, each stage’s Data availability genes were first subtracted from the previous stages’ DEGs and then tested for The datasets generated during and/or analysed during the current study are available in overlap with subsequent stages (Supplementary Fig. 3). GEO accession code number GSE134508. To assess the significance of added shared genes, we used rotation gene set testing functionality (ROAST)24 in the limma package59. ROAST can test whether any of the genes in a given set of added shared genes are differentially expressed in the specified Code availability contrast and also can test if they are consistently regulated. ROAST tests for three Codes are available on GitHub (https://github.com/bukhariabbas/stickleback-paternal-care). alternative hypotheses: “Up”, tests whether the genes in the set tend to be up- All other relevant data is available upon request. regulated, “Down” tests whether the genes in the set tend to be down-regulated and “Mixed” tests whether the genes in the set tend to be differentially without regard for Received: 7 December 2018 Accepted: 28 August 2019 direction of regulation. Here we used directional ROAST (null hypothesis either Up or Down) and separated the added shared genes by their direction of regulation (up or down) in a focal stage and then tested for their significant differential expression and consistent direction in subsequent stages. We also complemented this analysis with a Chi-Square test to determine whether the number of genes within a given set of overlapping genes showing a concordant expression pattern is greater than expected due to chance. References 1. Clutton-Brock, T. H. The Evolution of Parental Care. (Princeton University Press, 1991). Stickleback and mouse orthrogroups. To compare stickleback and mouse genes 2. Royle, N. J., Smiseth, P. T. & Kolliker, M. The Evolution of Parental Care. we generated a reliable orthogroup map using OrthoDB, v9.160. This map con- (Oxford University Press, 2012). tained both one-to-one, one to many and many to many orthology associations 3. DeAngelis, R. S. & Rhodes, J. S. Sex differences in steroid hormones and between stickleback and mouse genes. This map contains 3790 orthogroups which parental effort across the breeding cycle in Amphiprion ocellaris. Copeia 104, represent 4820 stickleback and 4894 mouse genes. 586–593 (2016).

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62. Chandrasekaran, S. et al. Behavior-specific changes in transcriptional modules Additional information lead to distinct and predictable neurogenomic states. Proc. Natl Acad. Sci. USA Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 108, 18020–18025 (2011). 019-12212-7. 63. Shpigler, H. Y. et al. Deep evolutionary conservation of autism-related genes. Proc. Natl Acad. Sci. USA 114, 9653–9658 (2017). Competing interests: The authors declare no competing interests. 64. Saul, M. C. et al. Transcriptional regulatory dynamics drive coordinated metabolic and neural response to social challenge in mice. Genome Res. 27, Reprints and permission information is available online at http://npg.nature.com/ 959–972 (2017). reprintsandpermissions/ 65. Mi, H. et al. PANTHER version 11: expanded annotation data from gene ontology and reactome pathways, and data analysis tool enhancements. Peer review information Nature Communications thanks the anonymous reviewer(s) for Nucleic Acids Res. 45, D183–d189 (2017). their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements We thank Gene Robinson, Mark Hauber, Dave Zhao, Saurabh Sinha, Lisa Stubbs, Mikus Abolins-Abols and members of the Bell lab for comments on the paper. Bukhari was Open Access This article is licensed under a Creative Commons supported by a Dissertation Improvement Grant from the University of Illinois during the Attribution 4.0 International License, which permits use, sharing, preparation of this paper. This material is based upon work supported by the National adaptation, distribution and reproduction in any medium or format, as long as you give Science Foundation under Grant No. IOS 1121980, by the National Institutes of Health appropriate credit to the original author(s) and the source, provide a link to the Creative under award number 2R01GM082937-06A1 and by a grant from the Simons Foundation to L. Stubbs and Gene Robinson. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory Author contributions regulation or exceeds the permitted use, you will need to obtain permission directly from S.A.B. contributed to study design, analyzed the data and wrote the first draft of the the copyright holder. To view a copy of this license, visit http://creativecommons.org/ paper. M.S. contributed to study design and data analysis. N.J., M.B., L.R.S. and R.T. licenses/by/4.0/. contributed to study design and collected the data. A.M.B. designed the study, con- tributed to data analysis and interpretation and edited the paper. All authors approved the final version of the paper. © The Author(s) 2019

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