
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

37292748
10.1101/2023.05.26.542531
preprint
4
Article
Activity of FoxP2-positive neurons is associated with tadpole begging behavior
Ludington Sarah C. Conceptualization Methodology Formal Analysis Investigation Data Curation Writing – original draft preparation Visualization
McKinney Jordan E. Investigation Writing – review and editing
Butler Julie M. Writing – review and editing Supervision
Goolsby Billie C. Methodology Investigation Visualization
Callan Ashlyn A. Investigation Visualization
Gaines-Richardson Maiah Investigation Writing – review and editing
http://orcid.org/0000-0002-2706-4077
O’Connell Lauren A. Conceptualization Formal Analysis Resources Writing – original draft preparation Supervision Project administration Funding acquisition *
Department of Biology, Stanford University, Stanford, CA 94305, USA
* To whom correspondence should be addressed: Lauren A. O’Connell, Department of Biology, Stanford University, 371 Jane Stanford Way, Stanford, CA 94305, loconnel@stanford.edu
29 8 2024
2023.05.26.542531https://creativecommons.org/licenses/by-nd/4.0/ This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
nihpp-2023.05.26.542531.pdf
Motor function is a critical aspect of social behavior in a wide range of taxa. The transcription factor FoxP2 is well studied in the context of vocal communication in humans, mice, and songbirds, but its role in regulating social behavior in other vertebrate taxa is unclear. We examined the distribution and activity of FoxP2-positive neurons in tadpoles of the mimic poison frog (Ranitomeya imitator). In this species, tadpoles are reared in isolated plant nurseries and are aggressive to other tadpoles. Mothers provide unfertilized egg meals to tadpoles that perform a begging display by vigorously vibrating back and forth. We found that FoxP2 is widely distributed in the tadpole brain and parallels the brain distribution in mammals, birds, and fishes. We then tested the hypothesis that FoxP2-positive neurons would have differential activity levels in begging or aggression contexts compared to non-social controls. We found that FoxP2-positive neurons showed increased activation in the striatum and cerebellum during begging and in the nucleus accumbens during aggression. Overall, these findings lay a foundation for testing the hypothesis that FoxP2 has a generalizable role in social behavior beyond vocal communication across terrestrial vertebrates.

amphibian
begging
aggression
social behavior
neural activity
striatum
cerebellum
National Institutes of HealthDP2HD102042 Rita Allen Foundation, Pew Charitable Trusts, The New York Stem Cell Foundation, Stanford University Biology Summer Undergraduate Research Program Fellowship, Stanford University Major
==== Body
pmcIntroduction

In species where parents provision their young, offspring signaling can be important for obtaining food. Begging behavior generally involves coordination of motor circuits, such as vocalization in chicks [1–3], vibrational displays in amphibian tadpoles [4,5], and chemical and motor signals in insect larvae [6,7]. There is a rich theoretical literature on the evolution of offspring signaling and parental investment [8,9], which has been experimentally investigated mostly in birds [10–12]. While the behavioral and physiological ecology of begging has received much attention, the neural basis of this critical behavior is relatively unknown, with the exception that bird begging behavior uses the same vocal-motor pathways later used in adult song [13]. Investigating the neural circuits and gene networks that regulate offspring signaling would establish a mechanistic view on how begging behavior evolves from ancestral neural features. Additionally, this perspective would complement the existing theoretical models of how and when begging signals evolve.

Forkhead Box P2 protein (FoxP2) is associated with motor processes related to behavior in many species. This protein is a transcription factor that regulates gene networks important in many neuronal functions, including genes involved in synaptic plasticity, neurotransmission, and axonal guidance [14,15]. Interest in FOXP2 surged when a mutation was linked to speech and language impairments in humans [16] (human FOXP2 and non-human FoxP2 homologs are upper and lowercase, respectively). The mutation of a critical residue in the DNA-binding domain of the human FOXP2 (the R553H mutation) causes difficulty with fine rapid movements of the mouth and face that impair speech [17]. FOXP2 truncations and intragenic deletions also manifest in language and speech impairments [18–20]. Individuals carrying FOXP2 disruptions are at risk for other phenotypes such as difficulties feeding in infancy and low performance in receptive and expressive language assessments [21]. A conserved role for FoxP2 has been extended to other species, where FoxP2 manipulations influence vocalizations emitted by birds [22–24] and mice [25–27]. Functional studies in humans, mice, and birds point to the role of FoxP2 in the development and function of corticostriatal and cortico-cerebellar circuits important for motor control [16,28,29]. Despite the research emphasis on vocal communication, FoxP2 manipulations in mice lead to altered social interactions, like parental care and aggression [30,31], as well as skilled motor tasks [32], suggesting a broad role for FoxP2 in coordinating motor aspects of behavior.

Frogs use both vocal and non-vocal signaling for social interactions [33,34], but the role of FoxP2 in frog communication has not yet been investigated, to our knowledge. Like in mammals [35], FoxP2 is expressed in early brain development of Xenopus [36]. Given expression of FoxP2 in the larval brain and that FoxP2 is important in regulating motor aspects of social behavior in mammals and birds, we reasoned that FoxP2 may play a role in amphibian social behaviors as well. Specifically, since FoxP2 has an important role in vocal-motor pathways of bird song [22–24], which are more active in begging birds [13], we reasoned that FoxP2 may be involved in tadpole begging behavior. Additionally, as FoxP2 also leads to altered aggression in mice [30], we reasoned that FoxP2 may also be associated with tadpole aggression. In the present study, we tested the hypothesis that FoxP2 is associated with begging signals of tadpoles towards adult conspecifics or aggression towards conspecifics. We tested this hypothesis in the mimic poison frog (Ranitomeya imitator), where tadpoles beg parents for unfertilized egg meals by vigorously vibrating back and forth with their heads and nipping at visiting females with their mouths [37]. Tadpoles are reared in isolated nurseries where they are aggressive to intruder tadpoles [4,38]. We first mapped the neural distribution of FoxP2 in the R. imitator tadpole brain and then compared the activity of FoxP2-positive neurons across begging, aggressive, and control animals. Given the extensive literature of striatal FoxP2 in vocal communication in birds and mice, we predicted that FoxP2-positive neurons in the striatum would have higher activity in begging tadpoles.

Methods

Animals

Ranitomeya imitator tadpoles were bred from our laboratory colony [39]. Adult R. imitator females from breeding pairs were used as stimulus animals in the begging context. A conspecific tadpole was used as a stimulus in the aggression context. All procedures were approved by the Stanford University Animal Care and Use Committee (Protocol #33097).

Behavior

We randomly assigned tadpoles (Gosner stage 30–34, no forelimb development and minimal hindlimb development) into one of three experimental groups: a reproductive adult female (begging, N=14); a smaller sized conspecific tadpole (aggression, N=15), or exposed to a novel object (a metal bolt, N=15). Conspecific stimuli were different between trials. All behavior trials were conducted between 09:00 and 12:00 hours. Tadpoles were placed into individual square arenas (5 × 5 × 5 cm) filled with 50 mL of conditioned water (Josh’s Frogs R/O Rx, Owosso, MI). Tadpoles were recorded from above using GoPro cameras (GoPro HERO7 Black, 1080p, 240 fps). Each tadpole acclimated for 10 min in the arena. Then, the stimulus was introduced to the arena and behavior was recorded for 30 min. Stimuli were then removed from the arena and tadpoles were placed in the dark for 15 minutes to minimize post stimulus neural activity. This additional time was included as pilot experiments with pS6-immunoreactivity suggests this marker peaks 45 min post stimulus. Tadpoles were then anesthetized with topical 20% benzocaine and euthanized by decapitation.

Videos were scored using BORIS software [40] by an observer uninformed of tadpole identity (Figure S1). Begging was quantified by the number and duration of each begging bout, where the tadpole orients to, intensely vibrates near, and occasionally nips at a conspecific. Aggression and cannibalism is observed in tadpoles of this species, where tadpoles will attack and consume conspecifics. In this study, aggression was quantified by the number and duration of attacks towards the other tadpole. Control tadpoles did not display either of these behaviors.

Immunohistochemistry

Whole tadpole heads were fixed with 4% paraformaldehyde (PFA) in 1X phosphate buffered saline (PBS) at 4°C overnight, rinsed in 1X PBS, and transferred to a 30% sucrose solution for cryoprotection at 4°C overnight. Samples were then embedded in mounting media (Tissue-Tek® O.C.T. Compound, Electron Microscopy Sciences, Hatfield, PA, USA) and stored at −80°C until cryosectioning at 15 μm into three series. Sections were thaw-mounted onto SuperFrost Plus microscope slides (VWR International, Randor, PA, USA) and then stored at −80°C until immunohistochemistry.

We used double-label fluorescence immunohistochemistry to detect FoxP2 and phosphorylated ribosomes (pS6, phospho-S6) as a proxy of neural activity [41], as previously described [42]. Slides were incubated overnight in a mix of both primary antibodies [rabbit anti-pS6 (Invitrogen, cat #44–923G) at 1:500 and goat anti-FoxP2 (Abcam, cat #AB1307) at 1:500 in 2% normal donkey, 0.3% TritonX-100, 1X PBS]. Following several washes, slides were incubated in a mix of fluorescent secondary antibodies (1:200 Alexa 488 donkey anti-goat and 1:200 Alexa 568 donkey anti-rabbit in 2% normal donkey serum, 0.3% TritonX-100, 1X PBS) for two hours. Slides were then rinsed in water and cover slipped using Vectashield Hardset Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and stored at 4°C. FoxP2 was restricted to cell nuclei and additional antibody characterization can be found in Supplementary Materials (Figure S2-S5).

Fluorescence microscopy and cell counting

Brain sections were imaged on a Leica compound fluorescent microscope with a QImaging Retiga 2000R camera as previously described [42]. Brain regions containing FoxP2 were identified using DAPI-stained nuclei while referencing a poison frog brain atlas [42]. FIJI software [43] was used to measure the area of the nucleus accumbens, striatum, and cerebellum within a single hemisphere. The number of FoxP2-positive cells, pS6-positive cells, and colocalized cells were quantified within each area using the “Cell Counter” function. Due to tissue quality, one to four sections were counted per individual per brain region.

Data analysis

All statistics and figures were generated in R Studio (version 1.1.442) running R (version 3.5.2). We used the glmmTMB R package [44] to analyze cell count data with generalized linear mixed models. For FoxP2-positive and pS6-FoxP2 colocalized cells, we ran separate models using a negative binomial distribution; model fit was confirmed using DHARMa [45]. For both models, we tested the main effects of the experimental group (begging, aggression, control), brain region, and their interaction. Tadpole identity was included as a random variable to account for repeated sampling of brain regions within individuals. The log of the brain region area was included as an offset. For colocalization data, the number of colocalized (pS6 + FoxP2) cells was the dependent variable and the number of FoxP2 cells was included as a weight in the model. We then used the Anova.glmmTMB function for reported statistical values. When there was a significant interaction between group and brain region, we ran a post-hoc test with the emmeans R package (version 1.5.3) and used false discovery rate correction for multiple hypothesis testing. Correlations between behavior and cell counts were tested using the cor.test function in the R base package with the Spearman method.

Results

Neural distribution of FoxP2

We observed a broad distribution of FoxP2-positive cells throughout the tadpole brain (Figure 1, Figure S6). The highest densities of FoxP2-positive cells were found in the subpallial forebrain, optic tectum, thalamus, and cerebellum. Notably, there were many FoxP2 cells in regions linked to sensory processing, such as the olfactory bulb (chemosensory), torus semicircularis (acoustic processing), and optic tectum (vision).

FoxP2-positive neuronal activity changes with different social stimuli

We investigated whether FoxP2-positive neuronal activity is associated with social behavior by quantifying the proportion of FoxP2-positive cells that colocalized with the pS6 marker of neural activity in tadpoles showing begging, aggression, or exposed to a novel object (asocial control) (Figure 2). We focused our quantification efforts on the basal ganglia (nucleus accumbens and striatum) and cerebellum given their robust expression of FoxP2 in mice and birds [46] and functional studies suggesting FoxP2-associated vocalization deficits are due to altered corticostriatal and corticocerebellar circuits [16,28,29]. The activity of FoxP2-positive cells depended on an interaction of behavioral group and brain region (group*region: F4 = 130.66, p < 0.001). Begging tadpoles had more active FoxP2-positive cells than aggressive and control tadpoles in the striatum (Str, aggression vs begging: z = −3.144, p = 0.005; begging vs control: z = 2.517, p = 0.018) and cerebellum (Cb, aggression vs begging: z = −2.490, p = 0.019; begging vs control: z = 3.626, p < 0.001). The number of active FoxP2-positive cells did not differ between aggressive and control animals in the striatum (p = 0.175) or cerebellum (p = 0.920). Aggressive tadpoles had more active FoxP2-positive cells than control tadpoles in the nucleus accumbens (NAcc, z = 2.989, p = 0.008), whereas activity of FoxP2-positive cells in this brain region did not differ between begging and aggression (p = 0.125) or control (p = 0.125) contexts. There was a significant difference in the number of FoxP2-positive cells within these brain regions across groups, where aggressive tadpoles had more FoxP2-positive cells in the striatum (Figure S7). There were no significant differences in the number of pS6 cells within these brain regions across groups and there were no significant correlations between activity of FoxP2-positive cells and measures of begging or aggressive behaviors (Figure S8).

Discussion

Across species, there is variation in whether and how young animals display aggression or signaling to caregivers, but both behaviors require motor function coordinated by neural processes. Among other functions, the transcription factor FoxP2 plays a well established role in coordinating social behaviors like vocal communication in mammals and birds [17,20,24,28,47,48]. Our study expands the role of FoxP2 to social behavior in amphibians, laying a foundation for testing the generalizable function of FoxP2 in coordinating aspects of social behavior across terrestrial vertebrates in future studies.

The brain distribution of FoxP2 is conserved across vertebrates

FoxP2 is widespread throughout the amphibian brain, with a distribution pattern consistent with those found in other vertebrates (mammals: [35,49,50]; birds: [51]; fish: [52–54]). Across these taxa, there is a conserved pattern of expression in brain areas involved in motor output, sensory processing, and sensorimotor integration. In R. imitator tadpoles, brain regions that regulate motor output and social behavior, including the basal ganglia and cerebellum, had many FoxP2-positive cells. FoxP2 is expressed in the basal ganglia and cerebellum in avian vocal and non-vocal learners, crocodiles, and rodents, suggesting conserved expression in motor-related areas regardless of the ability to learn acoustic communication [49,51]. Although there is conservation in the presence of FoxP2, its abundance is variable in songbirds depending on age and environment, suggesting FoxP2 expression may be linked to periods of vocal plasticity [51]. We also noted FoxP2-positive cells in many sensory processing regions like the olfactory bulb (chemosensory), optic tectum (visual processing), and torus semicircularis (acoustic processing). In bats, species differences in FoxP2 expression in the olfactory bulb are associated with different feeding habits (frugivorous versus insectivorous) [55], suggesting that FoxP2 may influence olfactory processing. Given R. imitator tadpoles rely on smell to distinguish between heterospecific stimuli [39], investigating FoxP2’s role in sensory integration broadly may be a valuable future research direction. However, the expression pattern of FoxP2 is variable across sex, age, and species [49,56], including species differences in expression of FoxP2 in neuronal and non-neuronal cells in the mammalian cortex [57]. This variability in expression makes the regulatory role of FoxP2 in behavior unclear, but a fruitful avenue of future research. Overall, the distribution of FoxP2 in the amphibian brain suggests a largely conserved pattern across terrestrial vertebrates.

A general role for FoxP2 in social behavior

We found that activity of FoxP2-positive neurons was higher in the striatum and cerebellum of begging tadpoles and in the nucleus accumbens of aggressive tadpoles. Whether this pattern is directly relevant to these behaviors requires functional manipulations in a brain region-specific manner. Regardless, the context-dependent neuronal activation points to brain region specific roles for FoxP2-positive cells in social behavior. These findings lay a foundation for testing the hypothesis that FoxP2 has a generalizable role in social behavior beyond vocal communication.

The striatum is important for motor skills in many vertebrates [58] and has been linked to vocal communication in several taxa [59]. We found that FoxP2-positive cells in the striatum have increased activity during tadpole begging, suggesting a function for this brain region in tadpole signaling. This is supported by many studies regarding the role of FoxP2 in the striatum of vocalizing birds and mammals. Deficits in songbird vocalizations are observed after FoxP2 knockdown in Area X, a striatal nucleus involved in song learning [24]. At a cellular level, FoxP2 has been implicated in structural plasticity, where FoxP2 modifications influence spiny dynamics of Area X neurons in zebra finches [60] and dendrite lengths of striatal neurons in mice [61]. In this same study, the variant of FoxP2 expressed in these mice also impacted dopamine concentrations in the striatum and nucleus accumbens. Dopamine signaling is critical to tadpole begging behavior [39], and our results here suggest a potential role for FoxP2 in dopamine signaling that should be investigated in the future. This general cellular dysregulation can be seen in mice with FoxP2 mutations, where the striatum is more active and motor-skill learning is disrupted due to abnormal temporal coordination of striatal firing [62]. Together, our work expands the potential role of FoxP2 in the striatum to behavioral signaling in amphibians, suggesting a conserved role for striatal FoxP2 in communication across tetrapod vertebrates.

The cerebellum is a highly conserved vertebrate brain region that coordinates voluntary movements and motor learning [63]. The cerebellum is also implicated in language [64], as there is higher overall cerebellar activity during language tasks in humans [65]. Mice expressing the FoxP2 with the R552H mutation (that leads to speech-language disorders in humans) have impaired ultrasonic vocalizations and poor dendritic development of FoxP2-positive cerebellar Purkinje cells [46]. A reduction of FoxP2 expression specifically in cerebellar Purkinje neurons leads to a reduction of ultrasonic vocalizations in mouse pups [66]. Moreover, expressing the wild type human FOXP2 in the cerebellum partially rescues ultrasonic vocalizations in mice with global expression of FoxP2 with the R552H mutation [67]. To our knowledge, the role of FoxP2 in the cerebellum during vocal learning in songbirds is unknown, but cerebellar lesions impair song learning [68]. Our study, along with studies in neonatal mice, suggest that investigating the role of cerebellar FoxP2 during vocal signaling in songbirds would resolve whether the role of these neurons in coordinating motor signaling are generalizable across taxa.

The nucleus accumbens brain region in the basal ganglia involved in motivation and behavioral reinforcement. In this study, we found increased colocalization of pS6 and FoxP2 in the nucleus accumbens of aggressive tadpoles compared to controls. In mice, increased neural activation in the nucleus accumbens is observed with aggression seeking behavior [69]. Only one study, to our knowledge, has examined the role of FoxP2 specifically in the nucleus accumbens, where deletion in adult mice leads to altered reward and fear learning ([70]; this study did not report effects on aggression). Heterozygous FoxP2+/− mice also show altered aggression in resident-intruder and maternal aggression assays, although the brain regions regulating these altered behaviors was not studied [31]. Our data suggests investigating nucleus accumbens FoxP2 function in the context of aggression would be a fruitful avenue of future research.

Summary

We present evidence that FoxP2 has conserved brain expression patterns across vertebrates by filling in a critical taxonomic gap from amphibians. We also show that parent-directed signaling by tadpoles is associated with activity of FoxP2-positive cells in the striatum and cerebellum. In contrast, activity of FoxP2-positive cells in the nucleus accumbens was associated with aggression. Overall, this work supports the hypothesis that the FoxP2 transcription factor is part of a molecular toolkit important for social behavior via striatal and cerebellar circuits across many animals.

Supplementary Material

Supplement 1

Supplement 2

Supplement 3

Acknowledgements

We thank Madison Lacey and David Ramirez for their support maintaining our poison frog colony. We especially appreciate comments on early versions of this manuscript from Neil Khosla. We acknowledge that our work takes place on the ancestral and unceded land of the Muwekma Ohlone tribe.

Funding

This work was supported by the National Institutes of Health [DP2HD102042], the Rita Allen Foundation, Pew Charitable Trusts, and The New York Stem Cell Foundation to LAO. LAO is a New York Stem Cell Foundation – Robertson Investigator. SCL and JEM were supported by a Stanford University Biology Summer Undergraduate Research Program Fellowship and SCL was supported by a Stanford University Major Grant.

Data Accessibility

All data are included in supplementary materials.

Figure 1. Neural distribution of FoxP2 in amphibians is similar to other vertebrates.

FoxP2 is widely distributed throughout the amphibian brain, including the subpallial forebrain (A), midbrain (B), and a few hindbrain regions (C). The center sagittal brain schematic (rostral is to the left) shows brain regions (green) with FoxP2 positive cells. Gray boxes represent areas of interest for more detailed neuroanatomy and micrographs (A1-C1), where green dots represent qualitative presence of FoxP2. Micrographs show FoxP2-positive cells (cyan) and DAPI-stained nuclei (blue); scale bar is 20 μm. The complete neural distribution for FoxP2 can be found in Supplementary Materials. Abbreviations: AMY, amygdala; aPOA, anterior preoptic area; BST, bed nucleus of the stria terminalis; Cb, cerebellum; Gc, central gray; Ls, lateral septum; mPOA, magnocellular preoptic area; NAcc, nucleus accumbens; OB, olfactory bulb; OT, optic tectum; SCN, suprachiasmatic nucleus; Str, striatum; VH ventral hypothalamus.

Figure 2. Activity of FoxP2 neurons changes with social behavior.

(A) Proportion of active FoxP2-positive cells in aggressive (orange), begging (dark green) and control (light green) tadpoles are shown in boxplots with individual tadpoles displayed in dots. Within each brain region, groups not connected by the same letter are significantly different. (B) Representative micrographs of FoxP2 (green) and pS6 (pink) colocalization expression of begging (left) or control (right) tadpoles in the striatum and cerebellum. Scale bar is 10 microns. Abbreviations: Cb, cerebellum; NAcc, nucleus accumbens; Str, striatum.
==== Refs
References

1. Rosenblatt JS . 2003 Outline of the evolution of behavioral and nonbehavioral patterns of parental care among the vertebrates: critical characteristics of mammalian and avian parental behavior. Scand. J. Psychol. 44 , 265–271.12914590
2. Mock DW , Dugas MB , Strickler SA . 2011 Honest begging: expanding from Signal of Need. Behav. Ecol. 22 , 909–917.
3. Budden AE , Wright J . 2001 Begging in nestling birds. In Current Ornithology, Volume 16 , pp. 83–118. Boston, MA: Springer US.
4. Yoshioka M , Meeks C , Summers K . 2016 Evidence for begging as an honest signal of offspring need in the biparental mimic poison frog. Anim. Behav. 113 , 1–11.
5. Stynoski JL , Stynoski PB , Noble VR . 2018 Empirical evidence for multiple costs of begging in poison frog tadpoles. Zool. Anz. 273 , 203–209.
6. Smiseth PT , Moore AJ . 2007 Signalling of hunger by senior and junior larvae in asynchronous broods of a burying beetle. Anim. Behav. 74 , 699–705.
7. Creemers B , Billen J , Gobin B . 2003 Larval begging behaviour in the antMyrmica rubra. Ethol. Ecol. Evol. 15 , 261–272.
8. Trivers RL . 1974 Parent-Offspring Conflict. Am. Zool. 14 , 249–264.
9. Godfray HC . 1995 Evolutionary theory of parent-offspring conflict. Nature 376 , 133–138.7603563
10. Hinde CA , Johnstone RA , Kilner RM . 2010 Parent-offspring conflict and coadaptation. Science 327 , 1373–1376.20223985
11. Dearborn DC . 1998 Begging behavior and food acquisition by brown-headed cowbird nestlings. Behav. Ecol. Sociobiol. 43 , 259–270.
12. McCarty JP . 1996 The energetic cost of begging in nestling passerines. Auk 113 , 178–188.
13. Liu W-C , Rivers JW , White DJ . 2016 Vocal matching and intensity of begging calls are associated with a forebrain song circuit in a generalist brood parasite. Dev. Neurobiol. 76 , 615–625.26335154
14. Vernes SC , Spiteri E , Nicod J , Groszer M , Taylor JM , Davies KE , Geschwind DH , Fisher SE . 2007 High-throughput analysis of promoter occupancy reveals direct neural targets of FOXP2, a gene mutated in speech and language disorders. Am. J. Hum. Genet. 81 , 1232–1250.17999362
15. Vernes SC 2011 Foxp2 regulates gene networks implicated in neurite outgrowth in the developing brain. PLoS Genet. 7 , e1002145.21765815
16. Vargha-Khadem F , Gadian DG , Copp A , Mishkin M . 2005 FOXP2 and the neuroanatomy of speech and language. Nat. Rev. Neurosci. 6 , 131–138.15685218
17. Lai CS , Fisher SE , Hurst JA , Vargha-Khadem F , Monaco AP . 2001 A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413 , 519–523.11586359
18. Reuter MS 2017 FOXP2 variants in 14 individuals with developmental speech and language disorders broaden the mutational and clinical spectrum. J. Med. Genet. 54 , 64–72.27572252
19. MacDermot KD 2005 Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits. Am. J. Hum. Genet. 76 , 1074–1080.15877281
20. Turner SJ , Hildebrand MS , Block S , Damiano J , Fahey M , Reilly S , Bahlo M , Scheffer IE , Morgan AT . 2013 Small intragenic deletion in FOXP2 associated with childhood apraxia of speech and dysarthria. Am. J. Med. Genet. A 161A , 2321–2326.23918746
21. Morison LD 2023 In-depth characterisation of a cohort of individuals with missense and loss-of-function variants disrupting. J. Med. Genet. 60 , 597–607.36328423
22. Day NF , Hobbs TG , Heston JB , White SA . 2019 Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood. eNeuro 6 . (doi:10.1523/ENEURO.0071-19.2019)
23. Murugan M , Harward S , Scharff C , Mooney R . 2013 Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability. Neuron 80 , 1464–1476.24268418
24. Haesler S , Rochefort C , Georgi B , Licznerski P , Osten P , Scharff C . 2007 Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol. 5 , e321.18052609
25. Chabout J , Sarkar A , Patel SR , Radden T , Dunson DB , Fisher SE , Jarvis ED . 2016 A Foxp2 Mutation Implicated in Human Speech Deficits Alters Sequencing of Ultrasonic Vocalizations in Adult Male Mice. Front. Behav. Neurosci. 10 , 197.27812326
26. Castellucci GA , McGinley MJ , McCormick DA . 2016 Knockout of Foxp2 disrupts vocal development in mice. Sci. Rep. 6 , 23305.26980647
27. Shu W 2005 Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene. Proc. Natl. Acad. Sci. U. S. A. 102 , 9643–9648.15983371
28. Lai CSL , Gerrelli D , Monaco AP , Fisher SE , Copp AJ . 2003 FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder. Brain 126 , 2455–2462.12876151
29. Teramitsu I , Kudo LC , London SE , Geschwind DH , White SA . 2004 Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction. J. Neurosci. 24 , 3152–3163.15056695
30. Medvedeva VP 2019 Altered social behavior in mice carrying a cortical Foxp2 deletion. Hum. Mol. Genet. 28 , 701–717.30357341
31. Herrero MJ , Wang L , Hernandez-Pineda D , Banerjee P , Matos HY , Goodrich M , Panigrahi A , Smith NA , Corbin JG . 2021 Sex-Specific Social Behavior and Amygdala Proteomic Deficits in Mutant Mice. Front. Behav. Neurosci. 15 , 706079.34421555
32. French CA , Vinueza Veloz MF , Zhou K , Peter S , Fisher SE , Costa RM , De Zeeuw CI . 2019 Differential effects of Foxp2 disruption in distinct motor circuits. Mol. Psychiatry 24 , 447–462.30108312
33. Narins PM , Feng AS , Fay RR . 2006 Hearing and Sound Communication in Amphibians. Springer Science & Business Media.
34. Kelley DB . 2004 Vocal communication in frogs. Curr. Opin. Neurobiol. 14 , 751–757.15582379
35. Ferland RJ , Cherry TJ , Preware PO , Morrisey EE , Walsh CA . 2003 Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J. Comp. Neurol. 460 , 266–279.12687690
36. Schön C , Wochnik A , Rössner A , Donow C , Knöchel W . 2006 The FoxP subclass in Xenopus laevis development. Dev. Genes Evol. 216 , 641–646.16609867
37. Brown JL , Morales V , Summers K . 2010 A key ecological trait drove the evolution of biparental care and monogamy in an amphibian. Am. Nat. 175 , 436–446.20180700
38. McKinney JE , Ludington SC , Butler JM , O’Connell LA . 2022 Proopiomelanocortin (POMC) is a negative regulator of tadpole aggression through opioid receptor signaling. bioRxiv. (doi:10.1101/2022.11.28.518266)
39. Butler JM , Singh D , Baker P , Edwards SV , Summers K , O’Connell LA . 2023 Dopamine neurons govern olfactory-gated infant begging behavior. bioRxiv (doi:10.1101/2023.03.18.533277)
40. Friard O , Gamba M . 2016 BORIS : a free, versatile open‐source event‐logging software for video/audio coding and live observations. Methods Ecol. Evol. 7 , 1325–1330.
41. Knight ZA , Tan K , Birsoy K , Schmidt S , Garrison JL , Wysocki RW , Emiliano A , Ekstrand MI , Friedman JM . 2012 Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 151 , 1126–1137.23178128
42. Fischer EK , Roland AB , Moskowitz NA , Tapia EE , Summers K , Coloma LA , O’Connell LA . 2019 The neural basis of tadpole transport in poison frogs. Proc. Biol. Sci. 286 , 20191084.31311480
43. Schindelin J 2012 Fiji: an open-source platform for biological-image analysis. Nat. Methods 9 , 676–682.22743772
44. Brooks M 2017 glmmTMB Balances Speed and Flexibility Among Packages for Zero-inflated Generalized Linear Mixed Modeling. The R Journal. 9 , 378. (doi:10.32614/rj-2017-066)
45. Hartig F . In press. DHARMa: residual diagnostics for hierarchical (multi-level/mixed) regression models. R package version 0.3
46. Fujita E , Tanabe Y , Shiota A , Ueda M , Suwa K , Momoi MY , Momoi T . 2008 Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells. Proc. Natl. Acad. Sci. U. S. A. 105 , 3117–3122.18287060
47. Callaway E . 2011 ‘Language gene’ speeds learning. Nature Publishing Group UK. (doi:10.1038/nature.2011.9395)
48. Bowers JM , Perez-Pouchoulen M , Edwards NS , McCarthy MM . 2013 Foxp2 mediates sex differences in ultrasonic vocalization by rat pups and directs order of maternal retrieval. J. Neurosci. 33 , 3276–3283.23426656
49. Campbell P , Reep RL , Stoll ML , Ophir AG , Phelps SM . 2009 Conservation and diversity of Foxp2 expression in muroid rodents: functional implications. J. Comp. Neurol. 512 , 84–100.18972576
50. Rodenas-Cuadrado PM , Mengede J , Baas L , Devanna P , Schmid TA , Yartsev M , Firzlaff U , Vernes SC . 2018 Mapping the distribution of language related genes FoxP1, FoxP2, and CntnaP2 in the brains of vocal learning bat species. J. Comp. Neurol. 526 , 1235–1266.29297931
51. Haesler S , Wada K , Nshdejan A , Morrisey EE , Lints T , Jarvis ED , Scharff C . 2004 FoxP2 expression in avian vocal learners and non-learners. J. Neurosci. 24 , 3164–3175.15056696
52. Shah R , Medina-Martinez O , Chu L-F , Samaco RC , Jamrich M . 2006 Expression of FoxP2 during zebrafish development and in the adult brain. Int. J. Dev. Biol. 50 , 435–438.16525940
53. Itakura T 2008 The medaka FoxP2, a homologue of human language gene FOXP2, has a diverged structure and function. J. Biochem. 143 , 407–416.18079162
54. Pengra IGG , Marchaterre MA , Bass AH . 2018 FoxP2 Expression in a Highly Vocal Teleost Fish with Comparisons to Tetrapods. Brain Behav. Evol. 91 , 82–96.29672280
55. Chen Q , Wang L , Jones G , Metzner W , Xuan FJ , Yin J , Sun Y . 2013 FoxP2 and olfaction: divergence of FoxP2 expression in olfactory tubercle between different feeding habit bats. Acta Biol. Hung. 64 , 426–437.24275589
56. Thompson CK , Schwabe F , Schoof A , Mendoza E , Gampe J , Rochefort C , Scharff C . 2013 Young and intense: FoxP2 immunoreactivity in Area X varies with age, song stereotypy, and singing in male zebra finches. Front. Neural Circuits 7 , 24.23450800
57. Ma S 2022 Molecular and cellular evolution of the primate dorsolateral prefrontal cortex. Science 377 , eabo7257.36007006
58. Cataldi S , Stanley AT , Miniaci MC , Sulzer D . 2022 Interpreting the role of the striatum during multiple phases of motor learning. FEBS J. 289 , 2263–2281.33977645
59. Konopka G , Roberts TF . 2016 Animal Models of Speech and Vocal Communication Deficits Associated With Psychiatric Disorders. Biol. Psychiatry 79 , 53–61.26232298
60. Schulz SB , Haesler S , Scharff C , Rochefort C . 2010 Knockdown of FoxP2 alters spine density in Area X of the zebra finch. Genes Brain Behav. 9 , 732–740.20528955
61. Enard W 2009 A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell 137 , 961–971.19490899
62. French CA , Jin X , Campbell TG , Gerfen E , Groszer M , Fisher SE , Costa RM . 2012 An aetiological Foxp2 mutation causes aberrant striatal activity and alters plasticity during skill learning. Mol. Psychiatry 17 , 1077–1085.21876543
63. Sokolov AA , Miall RC , Ivry RB . 2017 The Cerebellum: Adaptive Prediction for Movement and Cognition. Trends Cogn. Sci. 21 , 313–332.28385461
64. Mariën P , Borgatti R . 2018 Language and the cerebellum. Handb. Clin. Neurol. 154 , 181–202.29903439
65. E K-H , Chen S-HA , Ho M-HR , Desmond JE . 2014 A meta-analysis of cerebellar contributions to higher cognition from PET and fMRI studies. Hum. Brain Mapp. 35 , 593–615.23125108
66. Usui N , Co M , Harper M , Rieger MA , Dougherty JD , Konopka G . 2017 Sumoylation of FOXP2 Regulates Motor Function and Vocal Communication Through Purkinje Cell Development. Biol. Psychiatry 81 , 220–230.27009683
67. Fujita-Jimbo E , Momoi T . 2014 Specific expression of FOXP2 in cerebellum improves ultrasonic vocalization in heterozygous but not in homozygous Foxp2 (R552H) knock-in pups. Neurosci. Lett. 566 , 162–166.24607928
68. Pidoux L , Le Blanc P , Levenes C , Leblois A . 2018 A subcortical circuit linking the cerebellum to the basal ganglia engaged in vocal learning. Elife 7 . (doi:10.7554/eLife.32167)
69. Golden SA , Jin M , Heins C , Venniro M , Michaelides M , Shaham Y . 2019 Nucleus Accumbens Drd1-Expressing Neurons Control Aggression Self-Administration and Aggression Seeking in Mice. J. Neurosci. 39 , 2482–2496.30655356
70. He B-H , Yang Y-H , Hsiao B-W , Lin W-T , Chuang Y-F , Chen S-Y , Liu F-C . 2024 Foxp2 Is Required for Nucleus Accumbens-mediated Multifaceted Limbic Function. Neuroscience 542 , 33–46.38354901
