Sociability genetically separable from social hierarchy in amniotes

Xin Lin; Guangyi Dai; Sumei Zhou; Yangyang Li; Ben Zhou; Yi-Hsuan Pan; Haipeng Li

doi:10.1016/j.isci.2025.112930

. 2025 Jun 18;28(7):112930. doi: 10.1016/j.isci.2025.112930

Sociability genetically separable from social hierarchy in amniotes

Xin Lin ¹, Guangyi Dai ¹, Sumei Zhou ¹, Yangyang Li ^1,², Ben Zhou ^1,³, Yi-Hsuan Pan ^2,³, Haipeng Li ^1,^3,^4,^∗

PMCID: PMC12270018 PMID: 40678513

Summary

Sociability and social hierarchy are two essential features to form a social group. However, it remains unknown whether sociability and social hierarchy are genetically separable. In this study, we examined the social hierarchy, social and social novelty preference of PAS1 knock-out and knock-in mice. PAS1 (placental-accelerated sequence 1) is an enhancer that modulates social hierarchy. We found that PAS1 knock-out mice lack social hierarchy while wallaby/chicken PAS1 knock-in mice establish stable social ranks. Moreover, social and social novelty preference was observed in all PAS1-mutant mice. PAS1 knock-in mice have stronger preference to interact with other mice than wild-type mice (C57BL/6). All PAS1-mutants have neither stress/aggression-like behavior nor olfactory impairment. Overall, our results showed that PAS1 is an indispensable regulatory element in the formation of social hierarchy while PAS1 regulates one of pathways modulating sociability. Therefore, sociability should be genetically separable from social hierarchy in amniotes.

Subject areas: Rodent behavior, Behavior genetics, Molecular biology

Graphical abstract

Highlights

•
PAS1, a social enhancer, is indispensable for amniotes to establish social hierarchy
•
PAS1 modulates sociability in amniotes
•
Sociability and social hierarchy are regulated differently in amniotes
•
The evolutionary process for the first emergence of social system

Rodent behavior; Behavior genetics; Molecular biology

Introduction

Sociability, the ability and tendency to have social interaction with others, is the basic and adaptive component of many species,¹ including human beings.²^,³ Social impairment and the lack of social interaction are considered the sign of multiple mental illnesses.⁴^,⁵ Another main characteristic of social animals is that the members of a group form different social levels, i.e., social hierarchy.⁶^,⁷ To form social hierarchy, animals have to sense not only environmental changes⁸^,⁹^,¹⁰^,¹¹ but also the social status of each individual in the group.¹²^,¹³ In groups with stable hierarchical relationships, dominant animals have more resources than subordinate individuals, such as larger territory,¹⁴ priority access to food, and more spouse options.¹⁵ Rank in the hierarchical system greatly affects the behavior of animals, including mating, attack, and stress responses.¹⁶ Then a stable social structure and organization can be established, which is crucial to the survival of the group.¹⁷ Therefore, the formation of social groups involves many developmental and neural factors, postnatal learning and memory.

It has been documented that the nonsocial-to-social and the social-to-nonsocial transition occurred multiple times during the evolution of amniotes.²^,¹⁸ Some amniotic species are solitary-living (nonsocial)¹⁹ while many amniotes are social.²⁰ The solitary-living indicates that an individual forages independently in its home range and encounters its mate only during the breeding season.²¹^,²² In social species, sociability is crucial for individuals to maintain the social hierarchy, mate choices and offspring nursing.²³ In nonsocial or solitary-living species, sociability is also important for individuals to seek their partners and for mothers to nurse their offspring although those species lack social hierarchy.¹⁸ Therefore, sociability is an extremely important trait for all amniotes while the nonsocial-to-social and the social-to-nonsocial transitions empower amniotes to adapt different environments. Since both of sociability and social hierarchy are coded in genomes, from an evolutionary perspective, sociability may be genetically independent of social hierarchy. However, this issue has never been investigated.

To address this important question, genetic components modulating sociability and social hierarchy warrant further investigation. Since a nonsocial-to-social transition occurred in the ancestral lineage of placental mammals,¹⁸^,²⁴^,²⁵ we recently developed a novel method Kung-Fu Panda to detect the accelerated evolution in both conserved and non-conserved genomic regions within the lineage.¹³ Using this approach, we identified a social enhancer PAS1 (placental-accelerated sequence 1), located upstream of the Lhx2 gene, a key transcription factor that regulates brain development.²⁶^,²⁷ To examine the function of PAS1, we first generated a knock-out mouse strain (PAS1^-/-). Since PAS1 was also found in marsupials and birds, we generated two knock-in mouse strains by using the PAS1 sequences of wallaby and chicken (PAS1^w/w: wallaby PAS1 knock-in mice; PAS1^c/c: chicken PAS1 knock-in mice). All the strains have the C57BL/6 background. PAS1 knock-in mice (PAS1^w/w and PAS1^c/c) are able to maintain stable social ranks in cages with mixed homozygotes and heterozygotes of mutant mice while stable social ranks cannot be established in PAS1 knock-out mice.¹³ Moreover, no structural abnormalities were found in the brain of PAS1-mutant mice. Overall, PAS1^-/- is the first mouse strain identified to lack social hierarchy and social ranks were demoted or promoted by mutating PAS1.

In this study, we examined the social rank stability and the sociability of the PAS1-mutant mice, focusing on the role of the enhancer PAS1 in modulating the two complex traits. We further examined aggression, home-cage, and olfactory related behaviors of these strains and explored whether PAS1-mutants increase anxiety and stress level. Our findings confirmed that PAS1 is an indispensable regulatory element in the formation of social hierarchy because PAS1 knock-out mice lack social hierarchy. We also found that PAS1 only regulates one of the pathways modulating sociability. Therefore, sociability and social hierarchy are genetically separable in amniotes.

Results

Stable/unstable social ranks of PAS1-mutants

To examine the social hierarchy of PAS1-mutant mice, we detected the social rank of caged homozygote PAS1-mutant mice and wild-type mice, using the social dominance tube test²⁸^,²⁹ (Figure 1A). Four male mice with the same genotype were housed together in each cage for at least eight weeks before the test (Table S1). It should be noted that we housed mice with the same genotype in one cage in this study. This was different from our previous study,¹³ in which mice with different genotypes were housed in the same cage to examine whether PAS1-alleles modulate the social rank.¹³ During the test, each mouse competed against its three cage mates to determine its social rank. When the ranks of four mice remained unchanged in one cage for at least four consecutive days, we considered their social ranks stable.

Stable/unstable social ranks of PAS1-mutant mice

(A) Schematic diagrams of the social dominance tube test and social ranks of caged mice. Stable linear: four mice in one cage have stable linear ranks; unstable: mice cannot gain stable ranks.

(B–E) Social ranks of male wild-type and PAS1-mutants. Homozygote male mice were caged.

(F–I) Social ranks of female wild-type and PAS1-mutants. Homozygote female mice were caged. The colors are assigned according to the ranks obtained on the last day. PAS1^m/m: wild-type mice; PAS1^w/w: wallaby PAS1 knock-in mice; PAS1^c/c: chicken PAS1 knock-in mice; PAS1^-/-: PAS1 knock-out mice. n: the number of cages tested.

PAS1 knock-in and knock-out mice showed varying abilities to establish stable social ranks. We found that all caged PAS1^w/w and PAS1^c/c male mice (wallaby and chicken PAS1 knock-ins) establish stable social ranks, similar to the patterns observed in caged wild-type ones (Figures 1B–1D). In contrast, caged PAS1^-/- male mice failed to establish stable social ranks (Figures 1E and S1), even after nearly 20 weeks of housing (Table S1). Similar results were observed in female mice cages (Figures 1F–1I and S2).

We observed no significant differences in time for experimental mice to gain stable ranks (Figures S3A and S3B) and no significant differences in group-housed time between the control and PAS1-mutants (Figures S3C and S3D; Tables S1 and S2). It was also unlikely that social ranks are associated with their body weight (Figures S3E and S3F). Moreover, we observed hair plucking in some cages. In the tube test, we found that all barbers are alpha mice (see Video S1). Thus, it demonstrated that the results of tube test correctly measure the social ranks of mice.

Video S1. Barber mice in social dominance tube test

In the tube test, we found that all barbers are alpha mice.

Download video file^{(3.6MB, mp4)}

Considering a nonsocial-to-social transition, we explored the evolutionary process for the first emergence of social system, represented by stable social ranks. We examined the association between stable social ranks and the number of PAS1^-/- individuals in one cage. Based on our previous study¹³ and the results aforementioned, we found that social ranks are unstable when there are four or two PAS1^-/- individuals in a cage. When the number of PAS1^-/- individuals was reduced to one in a cage, stable social ranks emerged (Figure S4B). When there were no PAS1^-/- individuals in a cage, and heterozygotes (PAS1^m/^-) and wild-type (PAS1^m/m) were equally mixed, we observed stable social ranks (Figure S4A). We also conducted social dominance tube test on male PAS1 knock-out heterozygotes (PAS1^m/^-), all of which established stable social ranks (Figure S4C). Therefore, during a nonsocial-to-social transition, the establishment of stable social ranks is likely determined by the number of homozygotes with non-functional PAS1 alleles, and the maximum number is one in each sub-population.

Sociability of PAS1-mutants

Since PAS1 knock-out homozygotes fail to form social hierarchy, it is important to examine the sociability of three PAS1-mutant strains. We investigated their social and social novelty preference using the three-chamber social test.³⁰^,³¹^,³²^,³³ During the social preference stage, there was one circular wire cup in the corner of left chamber that held a juvenile stranger wild-type mouse (stranger1) (Figure 2A), while another empty cup was left in the diagonal corner of right chamber. The male experimental wild-type mouse usually explores and sniffs around stranger1, demonstrating social preference (Figure 2B). We found that the total interaction time between experimental mice and stranger1 was significantly higher than that between the experimental mice and empty cup (two-way ANOVA, PAS1^m/m: p < 0.0001; PAS1^-/-: p < 0.0001; PAS1^w/w: p < 0.0001; PAS1^c/c: p < 0.0001) (Figure 2C). We also conducted the same test using female mice and the results were similar (two-way ANOVA, PAS1^m/m: p < 0.0001; PAS1^-/-: p < 0.0001; PAS1^w/w: p < 0.0001; PAS1^c/c: p < 0.0001) (Figure S2B). Consequently, all the tested mice including the wild-type and PAS1-mutants showed social preference.

Sociability of PAS1-mutant male mice

(A) Schematic diagram of social preference stage in three-chamber social test.

(B) Heatmap of mouse motion trail during the social preference stage.

(C) Investigation time of experimental mice toward empty stimuli and stranger1.

(D) Schematic diagram of social novelty preference stage.

(E) Heatmap of mouse motion trail during the social novelty preference stage.

(F) Investigation time of experimental mice toward social familiar (stranger1) and un-familiar (stranger2) mice during social novelty preference stage.

(G and H) Investigation time of experimental mice toward stimuli during the two stages. PAS1^m/m: n = 36; PAS1^-/-: n = 36; PAS1^w/w: n = 33; PAS1^c/c: n = 33 individuals. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns – not significant, by two-way ANOVA with Šídák’s multiple comparisons test (C and F), or by Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (G and H). Error bars represent standard deviation.

To examine whether PAS1 modulates sociability, we compared the total social interaction time between three PAS1-mutant strains and the wild-type. Three male PAS1-mutant strains, like the male wild-type, showed no-interest in empty cup (Figure 2G). However, compared to the wild-type, both of two PAS1 knock-in strains showed a significantly greater interest in stranger1 (Ordinary one-way ANOVA, PAS1^w/w: p = 0.015; PAS1^c/c: p = 0.0201) (Figure 2G), demonstrating that PAS1 modulates sociability. Moreover, we observed no difference in the interacting time between experimental mice and stranger1 when examining the wild-type and PAS1 knock-out strain. Overall, it was very likely that there are multiple pathways modulating sociability.

During the social novelty preference stage, the mouse used in the social preference stage (stranger1) remained in the left chamber while another stranger wild-type mouse (stranger2) was held in the right chamber (Figure 2D). We found that all male PAS1-mutants spend more time with stranger2 than with stranger1 (two-way ANOVA, PAS1^m/m: p < 0.0001; PAS1^-/-: p < 0.0001; PAS1^w/w: p < 0.0001; PAS1^c/c: p < 0.0001) (Figures 2E and 2F). We also confirmed this observation in female mice (PAS1^m/m: p < 0.0001; PAS1^-/-: p = 0.0002; PAS1^w/w: p < 0.0001; PAS1^c/c: p = 0.0009) (Figure S2C), indicating that PAS1-mutants have normal social memory and social novelty recognition. We then compared the interacting time between experimental mice and stranger1 and found that there is not significant difference between the wild-type and PAS1-mutants (Figure 2H). Similarly, when examining the interacting time between experimental mice and stranger2, there is not significant difference between the wild-type and PAS1 knock-out mice. However, the interacting time was significantly increased in both wallaby- and chicken-PAS1 knock-in mice, compared to the wild-type (Ordinary one-way ANOVA, PAS1^-/-: p = 0.9597; PAS1^w/w: p = 0.0027; PAS1^c/c: p = 0.0274). We also examined male PAS1 knock-out heterozygotes and found that PAS1^m/^- mice have normal social and social novelty preference (Paired t test: p = 0.001 and 0.0085) (Figure S4D). Overall, these results suggested that PAS1 modulates social novelty preference.

Dynamics of social and social novelty preference of PAS1-mutants

To further explore the dynamics of social and social novelty preference behavior in PAS1-mutant mice, an in-depth analysis of three-chamber social test results was performed. The sociability stage and the social novelty stage were partitioned into 5-second bins and the mean interaction time was obtained for each bin (Figure 3A). All experimental PAS1-mutant mice interacted more with unfamiliar stimulus (i.e., stranger1 during the sociability stage and stranger2 during the social novelty stage) at the beginning and then their social interest gradually decreased over time. This pattern was similar to which was observed in the wild-type. During the early exploratory phase (first three minutes) (Figure S5A) of the sociability stage, the interaction time of experimental mice with stranger1 was much longer than to the empty cup. In the late exploratory phase (last three minutes) (Figure S5B), wild-type and PAS1 knock-out mice showed diminished interest in stranger1 while wallaby- and chicken-PAS1 knock-in mice remained a high level of interest. Similar observations were obtained during the social novelty stage. We then compared the dynamics of social and social novelty preference between PAS1-mutant mice and the wild-type. The investigation behavior of each experimental mouse during total ten 2minutes of both stages was presented as heatmaps (Figure 3B). The long interactions were largely observed between unfamiliar stimulus and experimental mice. Overall, the wallaby- and chicken-PAS1 knock-in mice generally showed stronger interests in stranger mice than the wild-type mice, while all of them exhibited no interest in empty cup (Figure S5C).

Dynamics of social and social novelty preference of PAS1-mutant male mice

(A) Investigation time of experimental mice, averaged in every five seconds, during social and social novelty preference stages.

(B) Heatmap of the investigation behavior of experimental mice. Each row represents one experimental mouse. PAS1^m/m: n = 36; PAS1^-/-: n = 36; PAS1^w/w: n = 33; PAS1^c/c: n = 33 individuals.

Since social rank influences social behavior in mice,³⁴ we examined whether the social rank affects the social and social novelty preference (Figure S6). All the mice were grouped by their ranks if the social hierarchy was established. Naturally, PAS1 knock-out mice were excluded from this analysis because they failed to establish the social hierarchy. The results showed that mice of different ranks exhibited similar behavior in sociability (two-way ANOVA for each rank: p < 0.0001) and social novelty preference (two-way ANOVA for each rank: p < 0.0001) (Figures S6A and S6B). Additionally, the investigation time to strangers has no significant differences among different ranks (Figures S6C and S6D). Therefore, social rank does not affect social and social novelty preference behavior.

Stress/anxiety level and aggressive behavior of PAS1-mutants

To explore the physiological and behavioral changes of PAS1-mutant mice, elevated plus maze test³⁵^,³⁶ was used to assess their anxiety-like behavior³⁵^,³⁷ (Figure 4A). If a mouse spends more time in open arms than the control, it indicates that the mouse has lower anxiety level. All PAS1-mutants and the wild-type control mice showed no significant difference in the time of open arm exploration (Figures 4B and 4C), indicating that PAS1 does not modulate the anxiety-like behavior.

Anxiety behavior and aggressive behavior of PAS1-mutant mice

(A) Schematic diagram of elevated plus maze test, and the example of the mouse’s trajectory in an elevated plus maze test.

(B and C) The time spent in open arms and the speed of movement during the experiment period.

(D) Schematic diagram of open field test and exploring trails of PAS1^m/m and PAS1^-/- mice. (E and F) The time of central activities and the speed of movement during the experiment period.

(G) Schematic diagram of resident-intruder test.

(H and I) The latency to the first contact time and total investigation time of PAS1-mutant male mice during the resident-intruder test.

(J) Investigation time, averaged in every five seconds, during the resident-intruder test.

(B–F) Male mice used: n = 20, and female mice used: n = 8 individuals.

(H–J) PAS1^m/m: n = 13; PAS1^-/-: n = 14; PAS1^w/w: n = 11; PAS1^c/c: n = 14 individuals. ∗∗p < 0.01, ns – not significant, by Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent standard deviation.

We also used open field test to examine the anxiety level of PAS1-mutant mice (Figure 4D) by measuring their central activities. The more central activities, the lower anxiety level. We observed that PAS1^-/- male mice have more central activities than the wild-type, indicating that PAS1^-/- male mice may have a significantly reduced anxiety level (Ordinary one-way ANOVA, p = 0.0068) (Figure 4E). In other 11 comparisons, we observed no significant difference (Figures 4E and 4F). We further tested the level of plasma corticosterone of male mice, another stress-level indicator.³⁸^,³⁹^,⁴⁰ The results showed no significant differences among different genotypes (Table S3). Therefore, PAS1-mutants do not have an increased anxiety-like behavior.

Aggression is a common behavior in social animals.⁴¹ When a new social group is initially formed, social hierarchy is established through the process involving aggressive behaviors, such as chasing, attacking, pinning down, and aggressive grooming,⁴²^,⁴³ although C57BL/6 mice are generally less aggressive than other strains.⁴⁴^,⁴⁵^,⁴⁶ We then used the resident-intruder test to assess the aggressive behavior in PAS1-mutant mice (Figure 4G). For this test, an experimental male mouse was first separated from its cage mates and housed individually in a new cage for at least 24 h before the test. Then a juvenile intruder mouse was placed into the experimental mouse’s cage. To measure the level of aggressive behavior, we recorded the latency to the first active social contact of experimental mouse (as resident) and the total interaction time between experimental mouse and intruder mouse (Figures 4H and 4I). All PAS1-mutant and the wild-type mice showed similar behavior to intruders in the test. The results of in-depth analysis demonstrated that the social interest of experimental mice in the intruders decreased over time in the wild-type and the three PAS1-mutant strains (Figure 4J). Thus, the different PAS1-alleles are unlikely to alter the aggressive behavior in mice and the lack of social hierarchy in PAS1 knock-out mice is not due to the aggression alteration.

Nesting behavior was recorded to examine whether PAS1-mutant mice have the excessive stress or mental abnormalities, which served as one of the early indicators of behavioral defects.³⁶^,⁴⁷^,⁴⁸ There was no significant difference in the home-cage behavior among all PAS1-mutants, including eating, exploring, grooming, sleeping, and nesting (Figure S3G and Video S2). Their breeding ability was also normal because there was no significant difference in the number of offspring per litter among different strains (Figure S3H). Therefore, PAS1 does not substantially affect the stress/anxiety level, aggressive, and nesting behavior in mice.

Video S2. Home-cage behavior

There was no significant difference in the home-cage behavior among all PAS1-mutants, including eating, exploring, grooming, sleeping and nesting.

Download video file^{(6.5MB, mp4)}

Olfactory behavior of PAS1-mutant mice

The olfactory organs of mice affect many aspects of behavior, such as judging the environment, looking for food, marking territory, communicating with peers and mating.⁴⁹^,⁵⁰^,⁵¹ Considering that the Lhx2 gene affects the development of olfactory bulb,⁵² it is important to ensure its function of PAS1-mutant mice. To examine the olfactory behavior of PAS1-mutant mice, we conducted a food-seeking test.⁵³^,⁵⁴ We cut off the food supply for 24 hours before the experiment started. We then hid the food under clean bedding on the experiment day and recorded the latency time for mice to find the food (Figure 5A). Our results showed that there is no significant difference in food-seeking ability among PAS1-mutants (Figures 5B and 5C). Therefore, it is unlikely that PAS1-mutants suffer an olfactory impairment.

Odor novelty test has been proposed to assess the non-social recognition memory of an experimental mouse and determine whether it was attributable to olfactory impairment⁵³^,⁵⁵^,⁵⁶ (Figure 5D). We chose two odors that have been recommended⁵⁷^,⁵⁸ and recorded the exploration time to the new odor of the experimental mouse. Our results showed that all the control and PAS1-mutant mice recognize the two odors and show novelty preference (Figures 5E and 5F) (two-way ANOVA, male mice: PAS1^m/m: p = 0.0171; PAS1^-/-: p = 0.0074; PAS1^w/w: p = 0.0003; PAS1^c/c: p = 0.0074. Female mice: PAS1^m/m: p = 0.0005; PAS1^-/-: p < 0.0001; PAS1^w/w: p < 0.0001; PAS1^c/c: p < 0.0001). This novelty preference was also observed in PAS1 knocked-out heterozygotes (Paired t test: p = 0.0032) (Figure S4E). Moreover, we failed to find a significant difference in exploration time to the unfamiliar odor (Figures 5G and 5H). Therefore, we found that there is no olfactory impairment and PAS1-mutants have normal olfactory-related memory.

Discussion

Sociability and social hierarchy are strongly correlated, leading to the hypothesis that the regulatory mechanism of sociability and social hierarchy might be similar.⁴¹^,⁵⁹ However, in this study we found that the social enhancer PAS1 plays a key role in the formation of social hierarchy while PAS1 regulates one of sociability-related pathways (Figure 6). Therefore, we suggested that sociability and social hierarchy should be genetically regulated differently in amniotes.

A model illustrating how sociability is genetically separable from social hierarchy in amniotes

To measure the social dominance of mice, tube test is a binary interaction experiment and relatively less information can be obtained. There are other methods to measure the social dominance of mice, such as the Landau’s Modified h value,³⁴ visible burrow system,⁶⁰ barber test,⁶¹^,⁶² agonistic behavior test,⁶³ urine-marking test,¹⁶ and ultra-sonic vocalization assay.⁶⁴^,⁶⁵^,⁶⁶^,⁶⁷ However, considering that tube test is commonly used,⁷^,²⁸^,²⁹^,⁴³ and we observed that all barbers are alpha mice, similar to the previous findings,⁶¹^,⁶² we suggested that our results of tube test represent the social ranks of mice.

Since PAS1 is an enhancer, its regulatory activity stems from its transcription factors. To provide a preliminary clue about the differential regulatory activity, we used the JASPAR database⁶⁸ to assess potential differences in the transcription factors binding to mouse/wallaby/chicken PAS1s. We found that FOXO3 and En2 have different binding profiles to different PAS1. The two transcription factors are previously identified to be essential in sociability. Abnormal acetylation of FOXO3 may lead to impaired social and cognitive abilities,⁶⁹ and En2 mutants exhibit depressive-like behavior and social dysfunction.⁷⁰ Therefore, this preliminary analysis on transcription factors is consistent with our findings. However, it requires a serial of substantial works to reveal how transcription factors can differently regulate sociability and social hierarchy in amniotes.

Considering that PAS1 is one of enhancers of Lhx2 gene, and this gene has a high expression during the mouse embryonic brain and determines the development of central nervous system.²⁷ Loss of Lhx2 impairs pituitary development⁷¹^,⁷² and cortical formations due to premature neurogenesis and radial glia differentiation.⁷³ Lhx2 also regulates hippocampal neuron-glia fate,⁷⁴^,⁷⁵ thalamocortical connectivity,⁷⁶ and olfactory neuron development.⁷⁷^,⁷⁸ It is crucial for commissural tract formation⁷⁹ and amygdala patterning.⁸⁰^,⁸¹ During the past decades, various brain regions are found to involve in the regulation of social behavior, such as medial prefrontal cortex,³^,²⁸ nucleus accumbens,⁸² hippocampus,⁸³^,⁸⁴ amygdala,⁸⁵ and neuronal migration.⁸⁶ Thus, Lhx2 is one of key regulatory factors to determine the development of social brain. It requires substantial works to investigate whether different brain regions or neural circuits differently regulate sociability and social hierarchy in amniotes.

It is important to investigate sociability and social hierarchy related pathways. Various social behavior genes are also known, such as Dvl1,⁸⁷ Caspr2 (contactin-associated protein-like 2)⁸⁸ and Shank3.⁸⁹ Since PAS1 may influence sociability and social hierarchy through shared molecular pathways, challenges are expected how to distinguish the function of genes in the two pathways.

We then examined whether our finding can be extrapolated from mouse to amniotes. We previously found that human- and chicken-derived PAS1s differently enhance the expression of reporter gene in the spinal cord of mouse embryo.¹³ Strikingly, the same differentiated expression pattern was found using chicken embryo. Considering chicken and mouse diverged more than 300 million years ago, this indicates that the regulatory network of PAS1 is highly conserved in amniotes.¹³ Moreover, the social structure of chicken and wallaby was emerged because of two nonsocial-to-social transitions in different ancestral lineages. The mouse-, wallaby-, and chicken-PAS1s were found to have similar function in modulating sociability and social hierarchy. It provides supports that sociability may be genetically separable from social hierarchy in amniotes. However, more studies are still needed, such as the neural mechanisms underlying the social enhancer PAS1.

Since sociability is modulated by multiple pathways, genetic buffering and robustness is expected. This might be essential for the evolution of amniotes because genetic buffering and robustness ensures that the loss of function in a pathway, such as PAS1 knock-out, does not impair sociability. However, it remains to be further investigated how this redundancy has been maintained during the evolution although pleiotropy, the phenomenon of a single pathway influencing multiple traits, could be one of explanations.

There are multiple advantages for social hierarchy mainly regulated by one pathway. First, this feature provides evolutionary flexibility, which enables nonsocial-to-social transitions to occur multiple times in different lineages. Second, it is also prompted social-to-nonsocial transitions after social hierarchy has emerged. This explains why solitary-living mammals are frequently found. At last, this feature is ecologically important. Many top mammalian predators are solitary-living because each individual requires large territory, such as tigers and bears.⁹⁰ Therefore, the genetic separability between sociability and social hierarchy is essential for the evolution of amniotes.

It would be interesting to explore how social hierarchy emerges because nonsocial-to-social transitions occurred many times. Since PAS1 knock-out heterozygotes (PAS1^m/^-) has ability to establish stable social ranks, it demonstrated that a single copy of PAS1 allele is enough to cis-regulate downstream gene expression for the establishment of social hierarchy. Moreover, we found that the establishment of stable social ranks is determined by the number of homozygotes ( $\leq 1$ individual) with non-functional PAS1 alleles in each sub-population. These findings indicate that, to establish social hierarchy in a population, most of individuals should have the ability to collaborate with others. This provides an important clue about how social hierarchy emerges in the nonsocial-to-social transition.

It is expected that a few individuals carrying a newly derived PAS1 allele might have formed a group and established social hierarchy. The frequency of genotypes is a function of allele frequency, following the Hardy-Weinberg principle. After considering the random effect of small population, the estimated lower bound of the frequency of the newly derived PAS1 allele was 0.373–0.536 when social hierarchy first appeared in one group of an ancestral species by assuming that the group was composed of 10–20 diploid individuals (Figure S4F). Therefore, the newly derived PAS1 allele should not be rare when social hierarchy first emerged among sub-populations.

Different amniotic species, such as wolves, primates, and meerkats, have diverse social structures and organizations. In many species, alpha males/females always play a key role in the social dynamics of those wild populations.⁹¹^,⁹²^,⁹³^,⁹⁴ The replacement of dominant breeders is such an important event that affects the survival of these populations.⁹⁵ Therefore, we suggest that PAS1 and its downstream genes may have played roles in the determination of social structure and organization. When new beneficial mutations occurred on PAS1 and its downstream genes, these mutations should have got fixed quickly during the evolution of amniotes. These substitutions might alter the mechanisms to establish social hierarchy and subsequently affect the formation of social structure and organization. However, future works and large-scale experiments are needed to validate this hypothesis.

Social ranks of mice in a population have a significant and decisive impact on how individuals cope with depression and chronic stress.⁴^,⁹⁶^,⁹⁷ Changes in social rank and social behavior have important implications for their health effects. Social interaction disorders are the core symptoms of many mental illness states.⁴ It has been found that the loss of high social rank may lead to depression-like behavior in mice.⁹⁸ However, social ranks change frequently in PAS1 knock-out mice, and we failed to observe depression-like behavior. Therefore, PAS1-mutants may provide an opportunity to study the neural and molecular mechanism of anti-depression.

Overall, our study revealed that sociability is genetically separable from social hierarchy, which is essential for the evolution of amniotes. This finding evokes many important questions. Various brain regions have been confirmed to involve in the regulation of social behaviors in mice,³^,²¹^,²⁸^,⁹⁹^,¹⁰⁰ and it remains unclear in which brain regions and neural circuits PAS1 primarily acts. It is important to identify genes in PAS1-related pathways although a number of genes have been documented to influence social behaviors.⁸⁷^,¹⁰¹^,¹⁰² Additionally, it is essential to determine the developmental stages that are important for the functions of sociability and social hierarchy. These studies should help us to understand the nature of social structure/organization and provide new therapeutic target in mental-related diseases and stresses.

Limitations of the study

Our study demonstrates the important role of PAS1 in regulating sociability and social hierarchy. It is expected that PAS1 modulates these two processes through essential neural circuits and molecular pathways. However, the current data are not sufficient to identify these brain regions and molecular pathways. Additionally, transcription factors of PAS1 should play significant roles in separating sociability from social hierarchy although some key factors can be shared between the two processes. Overall, substantial efforts are urgently required to investigate these key issues comprehensively.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Haipeng Li (lihaipeng@sinh.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data reported in this paper are enclosed in the supplementary materials. This paper does not report original code. Any additional information should be available from the lead contact upon request.

Acknowledgments

We thank Dr. Feifan Guo and the animal caretaker team for assistance with animal care and experiments. This work was supported by the Eastern Talent Plan Leading Project, the National Natural Science Foundation of China, and the Chinese Academy of Sciences.

Author contributions

X.L., G.D., S.Z., Y.L., B.Z., Y.-H.P., and H.L. conceived the study. X.L., G.D., S.Z., and Y.YL. collected and analyzed the data. All authors discussed the results and wrote the paper.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Critical commercial assays

Mouse CORT ELISA Kit	Sangon Biotech	Cat# D721183
Chemicals
Thyme oil	MACKLIN	Cat# T921845
Lemon oil	MACKLIN	Cat# L922409

Experimental models: Organisms/strains

Mouse: C57BL/6	Shanghai Model Organisms Center, Inc	http://www.shmo.com.cn/English/
Mouse: C57BL/6_ΔmPAS1(PAS1 knock-out, PAS1^−/−)	Wang Y. et al.¹³	N/A
Mouse: C57BL/6_wPAS1 (wallaby-PAS1 knock-in, PAS1^w/w)
Mouse: C57BL/6_Cpas1 (chicken-PAS1 knock-in, PAS1^c/c)

Software

SuperMaze	Shanghai Xinruan Information Technology Co., LTD.	N/A
Prism 8	GraphPad Software	https://www.graphpad.com/scientific-software/prism/; RRID:SCR_002798

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Experimental model and study participant details

All animal experiments were performed in accordance with the protocols approved by the Committee and Laboratory Animal Department, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. Governmental and institutional ethical guidelines were followed. PAS1 knock-out and knock-ins were generated in our previous study.¹³ Generally, 9–24 weeks old adult mice were used for testing behaviors. Mice were housed in specific-pathogen-free condition.

Method details

Social dominance tube test

Four mice with same gender of the same genotype with similar age and weight were cage-housed together for at least two weeks before the test. The age difference of these mice in one cage was less than 10 days. During the test, mice were allowed to run through a transparent plexiglass tube of 40 cm in length and 3 cm in diameter for male/2.5cm in diameter for female, a size just sufficient to permit one adult mouse to pass through without reversing the direction. There is a small chamber (17 × 8 × 14 cm) at each end of the tube for temporary housing purpose.

To prepare for the test, each experimental mouse was given eight training runs every day for three days. Immediately before the test, experimental mice were given four additional training runs. During the test, two mice were released at each end of the tube. There is a moveable transparent plexiglass door in the middle of the tube to ensure that the two mice meet in the middle of the tube. If a mouse retreated from the tube within 2 min, it was given a score of zero. Another mouse that did not retreat was given a score of one. If no mice retreated within 2 min, the test was repeated. During the training stage, if no mice retreated in three successive trials, the test was carried out in the next day. These cases were rare and only observed in the early training stage of PAS1^−/− mice. Between trials, the tube was cleaned with 75% ethanol. From trial to trial, experimental mice were released at either end alternatively. For each pair of mice, two or three trials were conducted, and the mouse that won two times was considered the winner of the test. A new tube was employed for the next pair of mice. Mice were allowed to rest for at least 10 min between tests. Each mouse competed with its three cage-mates. To determine the stability of social ranks over time, mice were tested under the same conditions every day for at least seven days. The rank of an experimental mouse was assessed by the number of wins against its cage mates at the last day if the ranks remained stable for at least four consecutive days.

Three-chamber social test

Mice (between12-24 weeks old) with same gender were housed together according to standards and requirements for at least two weeks before the test.³³ The hand-made box (60 × 40 × 22cm) was divided into three chambers by clear plexiglass sheets with opening “door” to allow experimental mice to move between the chambers. A suitable white plexiglass sheet was used as the floor. Two identical circular wire cup-like containers, that are large enough to hold a single mouse, were used to hold stranger-mice. One was placed at the corner of the left chamber; the other was placed at the diagonal corner of the right chamber. The containers are comprised of metal wires to allow for air exchange and to prevent direct physical interactions between the inside animal (stranger) with the outside one (experimental mouse). In this study, 10–16 weeks old wild-type mice were used as strangers. All strangers were wild-type mice and purchased from the Southern Model Organisms to ensure that they were unfamiliar with experimental mice.

Before performing the test, a habituation phase was done. Experimental mouse was placed into a clean three-chamber social approach box with two empty cups and allowed to explore the box freely for 10 min. For the sociability stage, experimental mouse was first placed in the center chamber while the doors between chambers were closed. A juvenile stranger mouse (stranger1) was put in the wire cup of the left chamber, while the right cup was empty. And then the doors between chambers were opened to allow experimental mouse free access to explore three chambers for 10 min. For the sociability stage, a novel unfamiliar mouse was put in the previously empty cup of the right chamber, while the same familiar mouse used in the sociability stage (stranger1) was still in the left chamber. Then experimental mouse was allowed to free explore three chambers for another 10 min. After each trial, all chambers and wire cups were cleaned with 75% ethanol and air-dried for 10 min, to prevent olfactory cue bias and to ensure proper disinfection. Social behaviors were recorded and analyzed by using SuperMaze (Shanghai Xinruan Information Technology Co., Ltd.).

Elevated plus maze test

The elevated plus maze consisted of two open arms (25 × 5cm, with 0.5 cm tall ledges) and two closed arms (25 × 5cm, with 20 cm-high walls). The open arms and closed arms were extended from a central platform (5 × 5 cm) at 90° and the maze was elevated 75 cm above the floor. At the beginning of each test, the experimental mouse was placed in the central platform facing an open arm. The maze was cleaned with 75% alcohol after each test to avoid any olfactory traces. All of the tests were recorded with a camera mounted above the maze. The parameters, such as the number of times, resting time, and movement distance of the mice entering the open arm and the closed arm within five minutes, were counted using SuperMaze (Shanghai Xinruan Information Technology Co., Ltd.).

Open-field test

The open-field test was performed in a box (30 × 30 × 35 cm). At the beginning of each test, the experimental mouse was placed in the center of the box and activity was measured for 10 min. The box was cleaned with 75% alcohol after each trial. Mouse behavior was continuously videotaped by a camera located above the box. The open field was divided into the central area and peripheral area. The center area was 36% of the total area (a square of approximately 18 × 18 cm). The parameters, such as the number of entries into the center zone, the time spent in the center zone and the total distance traveled, were analyzed by SuperMaze (Shanghai Xinruan Information Technology Co., Ltd.).

Corticosterone

For the detection of total corticosterone in plasma, mouse CORT (Corticosterone) ELISA Kit (Sangon biotech, D721183-0096) was utilized following the manufacturer’s protocol. After anesthetizing the mice by isoflurane, cardiac blood collection was carried out, 0.5–0.8mL of blood was collected from each mouse, the blood was put into a centrifuge tube with EDTA, mixed well, and centrifuged at 1000 rpm for 15 min to obtain plasma. 50 μL of plasma and antibody were added to each well, then incubated, washed and chromogenic agent were added according to the specific steps of the instructions, and detected by a microplate reader.

Resident–intruder test

The day before the experiment, an experimental mouse (12–15 weeks old male mice) was separated from its cage mates and lived alone for one day. On the next day, the experimental mouse was placed in a testing room with its home cage for 15 minutes. During the active (dark) phase of the light/dark cycle, a marked intruder was placed into the experimental mouse’s home cage. Intruders (8–10 weeks old) with dark coats were identified with nontoxic red paint at the base of their tails. Repeated use of the same intruders was allowed, but at least 2h between uses was required. Mice were then left undisturbed and they were videotaped freely for 10 mins. The measures for the resident–intruder test of the latency to the first active social contact of resident and the total interaction time between resident and intruder mouse.

Food-seeking test

The food-seeking test was performed as described previously.⁵³^,⁵⁴ The food supply was cut off for 24 h before the test. A clean cage (30 × 35 × 20 cm) filled with a 3 cm depth of new bedding was used. A 2 g food pellet was buried 1.5 cm beneath leveled bedding in a randomly chosen corner of the cage. It was the same food that the mice were regularly fed with. At the beginning of test, the experimental mouse was placed in the opposite corner in regard to the pellet. Behavior was videotaped by a camera located above the cage until the mouse found the food pellet. Latency to find food was recorded. For each test, a clean cage, clean bedding, and new pellet of chow was used.

Odor novelty test

The odor novelty test was performed as described previously.⁵³^,⁵⁵^,⁵⁶ The same apparatus in the three-chamber social test was used. At the beginning of the test, a habituation phase was done. The experimental mouse was placed into a clean three-chamber social approach box with two empty cups and allowed to explore the box freely for 5 minutes. After the habituation session, a non-social object (a wooden cylinder, 5 cm in high, 2.5 cm in diameter) with 1 μL of lemon oil (Macklin, L922409) was introduced to the two wire cups of both chambers. The mouse was allowed to explore freely for five minutes. In the odor novelty trial, the wooden cylinder in the cup of right chamber was replaced by a new wooden cylinder with 1 μL of thyme oil (Macklin, T921845), and the mouse was allowed to explore for another five minutes. After each trial, all chambers and wire cups were cleaned with 75% ethanol and air-dried for 10 minutes, to prevent olfactory cue bias. Mouse behaviors were recorded and analyzed by using SuperMaze (Shanghai Xinruan Information Technology Co., Ltd).

Home-cage and nesting behavior

Four mice with same gender were housed together according to standards and requirements. A piece of absorbent cotton (10 × 8 × 0.5cm) was provided as nesting material in the cage. Pictures of the nests were taken after 48h co-housing to measure the ability of nesting. Mouse home-cage behavior was videotaped by a camera located in front of the cage.

Lower bound of PAS1 allele frequency to establish social hierarchy

Denote the ancestral non-functional allele of PAS1 by “A”, and the new PAS1 functional allele by “a”. Their frequency was $f (A) = p$ and $f (a) = q$ . We assumed that the “A” allele, such as the PAS1-null allele, has no function to establish social hierarchy. There were three genotypes in the ancestral species: AA, Aa, and aa. Their frequency was $p^{2}$ , $2 p q$ , and $q^{2}$ , respectively, following the Hardy-Weinberg principle. To establish social hierarchy, the genotypes of individuals in a group had to be Aa or aa. The maximum number of AA individual is 1. Therefore, the probability to form such a group is ${(1 - p^{2})}^{n} + n {(1 - p^{2})}^{n - 1} p^{2}$ , by considering random sampling effects, where n is the number of individuals in the group. If we assumed $n = 20$ and required that this probability is larger than 0.05, we obtained $f (A A) \leq 0.215$ , indicating that the lower bound of frequency of new PAS1 functional allele $f (a)$ is 0.536 (Figure S3F). We also obtained the lower bound of $f (a)$ to be 0.373 when $n = 10$ . Overall, these results indicated that the frequency of new PAS1 functional allele should not be low when social hierarchy first established in a group during a nonsocial-to-social transition.

Quantification and statistical analysis

Statistical analyses were performed using Prism 8 (GraphPad Software). Values are presented as mean ± SD. All data were first tested with Shapiro-Wilk normality test for the normal distribution. To compare behaviors between two groups in three-chamber social test and odor novelty test, p-values were calculated using the two-way ANOVA followed by the post hoc test with Šídák multiple comparisons test. For comparison of four groups with single independent variable in the resident-intruder test and three-chamber social test, as well as elevated plus maze test and open-field test, in addition to corticosterone concentrations test and also odor novelty test, p-values were calculated using the Ordinary one-way ANOVA followed by the post hoc test with Dunnett’s multiple comparisons. To analyze the single variable associated with the four ranks, p-values were calculated with RM one-way ANVOA followed by the post hoc test with Tukey’s multiple comparisons.

Published: June 18, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.112930.

Supplemental information

Document S1. Figures S1–S6 and Tables S1–S3

mmc1.pdf^{(1.9MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Barber mice in social dominance tube test

In the tube test, we found that all barbers are alpha mice.

Download video file^{(3.6MB, mp4)}

Video S2. Home-cage behavior

There was no significant difference in the home-cage behavior among all PAS1-mutants, including eating, exploring, grooming, sleeping and nesting.

Download video file^{(6.5MB, mp4)}

Document S1. Figures S1–S6 and Tables S1–S3

mmc1.pdf^{(1.9MB, pdf)}

Data Availability Statement

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PERMALINK

Sociability genetically separable from social hierarchy in amniotes

Xin Lin

Guangyi Dai

Sumei Zhou

Yangyang Li

Ben Zhou

Yi-Hsuan Pan

Haipeng Li

Summary

Graphical abstract

Highlights

Introduction

Results

Stable/unstable social ranks of PAS1-mutants

Figure 1.

Sociability of PAS1-mutants

Figure 2.

Dynamics of social and social novelty preference of PAS1-mutants

Figure 3.

Stress/anxiety level and aggressive behavior of PAS1-mutants

Figure 4.

Olfactory behavior of PAS1-mutant mice

Figure 5.

Discussion

Figure 6.

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Method details

Social dominance tube test

Three-chamber social test

Elevated plus maze test

Open-field test

Corticosterone

Resident–intruder test

Food-seeking test

Odor novelty test

Home-cage and nesting behavior

Lower bound of PAS1 allele frequency to establish social hierarchy

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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