Early parental deprivation during primate infancy has a lifelong impact on gene expression in the male marmoset brain

Adverse early life experiences are well-established risk factors for neurological disorders later in life. However, the molecular mechanisms underlying the impact of adverse experiences on neurophysiological systems throughout life remain incompletely understood. Previous studies suggest that social attachment to parents in early development are indispensable for infants to grow into healthy adults. In situations where multiple offspring are born in a single birth in common marmosets, human hand-rearing is employed to ensure the survival of the offspring in captivity. However, hand-reared marmosets often exhibit behavioral abnormalities, including abnormal vocalizations, excessive attachment to the caretaker, and aggressive behavior. In this study, comprehensive transcriptome analyses were conducted on hippocampus tissues, a neuroanatomical region sensitive to social attachment, obtained from human hand-reared (N = 6) and parent-reared male marmosets (N = 5) at distinct developmental stages. Our analyses revealed consistent alterations in a subset of genes, including those related to neurodevelopmental diseases, across different developmental stages, indicating their continuous susceptibility to the effects of early parental deprivation. These findings highlight the dynamic nature of gene expression in response to early life experiences and suggest that the impact of early parental deprivation on gene expression may vary across different stages of development.

by their parents.After birth, the marmosets underwent either human hand-or parental rearing for a duration of 3 months (Fig. 1a).Subsequently, they were housed together with their age-matched peer.To categorize the marmosets based on age, those falling within the age range of 13 to 19 months were classified as Young Marmosets, while those within the age range of 88 to 164 months were designated as Aged Marmosets (Fig. 1b).The gene expression alterations observed in the Young Marmosets due to human hand-rearing in the initial 3 months post-birth were classified as short-term consequences.Conversely, the gene expression alterations observed in Aged Marmosets were characterized as long-term consequences.To account for the potential impact of different feeding patterns and nutritional compositions on brain development, monthly weight measurements were conducted on both human hand-reared and parent-reared marmosets starting from birth (Fig. 1c).The results showed slight differences in body weight at 4 months of age between the two groups.Marmosets that underwent human hand-rearing during the first 3 months after birth showed distinct variations in body weight compared to those that received parental rearing.However, after reaching 7 months of age, no significant differences in body weight were observed among the marmosets, suggesting that their nutritional needs were adequately met for normal brain development.
In this study, while we did not conduct specific quantitative behavioral analysis or vocal measurements, notable behaviors were observed.They exhibited increased aggression or demanding behavior towards caretakers, suggesting potential social and behavioral implications of early parental deprivation, as previously described 19,20 .Furthermore, their vocal development seemed to resemble that of an immature infant.These observations underscore the influence of early-life experiences on both social conduct and vocalization in human hand-reared marmosets, as elaborated earlier 20 .
Taken together, we suggested that while there may be initial differences in body weight due to variations in feeding patterns, the nutritional needs of human hand-reared marmosets can be sufficiently met for normal brain development.However, the observed behavioral and vocal immaturity in human hand-reared marmosets indicates potential long-term effects of early parental deprivation on social behavior and communication skills.

The pathway of neuroactive ligand-receptor interaction may play a significant role in the short-term consequences of early parental deprivation
To investigate the molecular mechanisms underlying the short-term consequences of early parental deprivation in human hand-reared marmosets, a detailed analysis of gene expression changes in hippocampus tissues was conducted.It is hypothesized that early parental deprivation can influence the neuronal gene expression program by changing persistent epigenetic modifications in the developing brain.To assess these changes, comprehensive transcriptome analyses were performed using the SurePrint microarray technique from Agilent Technology.Hippocampus tissues obtained from two marmosets reared by their parents and three marmosets that underwent human hand-rearing were utilized in the study.The selected marmosets, classified as young marmosets, were within the age range of 13 to 19 months.By comparing the gene expression profiles between the human handreared and parent-reared young marmosets, we identified 613 genes that exhibited significant differential expression in the hippocampus.Out of these genes, 425 were found to be significantly upregulated, and 188 genes were significantly downregulated in the human hand-reared marmosets (Fig. 2a).The criteria used for determining differential expression were fold change greater than 2.0 for upregulated genes and fold change less than -2.0 for downregulated genes, with a significance level of P < 0.05.These findings suggest that early parental deprivation can cause significant alterations in gene expression patterns in the hippocampus, potentially impacting various molecular processes and pathways involved in neuronal function and development.
To identify the biological processes associated with gene expression changes in early parental deprivation, we conducted functional annotation analysis using the DAVID (Database for Annotation, Visualization, and Integrated Discovery) tools 30,31 .Among the 425 genes that displayed upregulation in human hand-reared marmosets, two Gene Ontology (GO) clusters with high enrichment scores were identified (Table .1).The primary cluster, exhibiting the highest enrichment, was associated with the Neuroactive-Ligand Receptor Interaction Pathway.Additionally, Gene Set Enrichment Analysis (GSEA) 32,33 indicated a correlation between the upregulated genes in human hand-reared marmosets and the Neuroactive-Ligand Receptor Interaction Pathway (Fig. 2b).Within this pathway, GABA Receptor family (GABRR1, GABRG1, GABRA2, GABRA3, GABRB1), and cholinergic receptor family (CHRNA5, CHRNA3, CHRNA10, CHRM5) and OXTR showed notable upregulation in human hand-reared marmosets (Fig. 2c).Conversely, no GO clusters with significantly high enrichment scores were identified among the 188 genes displaying downregulation in human hand-reared marmosets.This finding suggests that the genes upregulated in human hand-reared marmosets are involved in neuroactive ligand-receptor interactions, which may play a significant role in the short-term consequences of early parental deprivation.

Identification of potential pathways and processes involved in the long-term consequences of early parental deprivation
To investigate the persistence of gene expression changes observed in human hand-reared young marmosets into adulthood, we conducted comprehensive gene expression analyses in marmosets ranging from 88 to 164 months of age.By comparing the gene expression profiles between these two age groups, we aimed to assess whether the expression changes associated with early parental deprivation were maintained or altered in adulthood.This analysis provides insights into the long-term consequences of early parental deprivation on gene expression patterns in the hippocampus and can help us understand the molecular mechanisms underlying the lasting effects of early-life experiences.By comparing the gene expression profiles between human hand-reared and parent-reared aged marmosets (between 88 to 164 months of age), we identified 567 genes that showed significant differential expression in the hippocampus.Among these genes, 381 genes were found to be significantly upregulated (fold change > 2.0, P < 0.05) in the human hand-reared marmosets, while 186 genes were significantly downregulated The pathway of neuroactive ligand-receptor interaction may play a significant role in the shortterm consequences of early parental deprivation.(a) A volcano plot was generated to visualize the results of the microarray analysis comparing human hand-reared young marmosets to parent-reared young marmosets.In the plot, 425 genes that exhibited upregulation in the human hand-reared marmosets were shown in red color (fold change > 2.0, P < 0.05).On the other hand, 188 genes that displayed downregulation were shown in blue color (fold change < −2.0, P < 0.05).(b) Gene Set Enrichment Analysis (GSEA) of the neuroactive-ligand receptor interaction pathway enriched in human hand-reared marmosets compared to parent-reared young marmosets.(c) Heat map was visualized neuroactive ligand-receptor interaction pathway genes.HRY handreared young marmoset, PRY parent-reared young marmoset, red higher expression, blue lower expression.www.nature.com/scientificreports/(fold change < − 2.0, P < 0.05) in the human hand-reared marmosets (Fig. 3a).Furthermore, we performed GSEA to gain insights into the biological processes associated with these gene expression changes in human hand-reared aged marmosets.The analysis revealed that the downregulated genes in human hand-reared marmosets were associated with three gene sets: BOQUEST STEM CELL UP, NABA ECM GLYCOPROTEINS, and WU CELL MIGRATION (Fig. 3b). Figure 3c illustrates the partially or completely overlapped genes among these three gene sets.Notably, within these gene sets, several genes are of particular interest.The EFEMP1 gene, associated with dementia 34 , exhibited downregulation in human hand-reared marmosets, suggesting a potential link between its expression and the observed cognitive effects 19 .Furthermore, the IGFBP3/6 and WNT5a genes, known for their involvement in neuronal differentiation and proliferation 35,36 , showed downregulation in human hand-reared marmosets.Additionally, the gene S100A4, which has a neuroprotective pro-survival effect on neurons during brain injury 37 , was identified among the downregulated genes.Its downregulation suggests a potential reduction in the neuroprotective response, potentially contributing to increased vulnerability to adverse effects following early parental deprivation.Conversely, GSEA did not reveal any significant correlations between the upregulated genes in human hand-reared marmosets and the potential pathway related to behavior or neural development.These findings provide valuable insights into the molecular mechanisms underlying the consequences of early parental deprivation in human hand-reared aged marmosets.The identified gene sets and associated genes shed light on the potential pathways and processes involved in the observed gene expression changes and associated cognitive and behavioral alterations.

Identification of genes continuously affected by early parental deprivation throughout life
In this study, we delved into the gene expression profiles within the hippocampus of both young and aged human hand-reared marmosets, comparing them to their parent-reared counterparts.Our objective was to understand the persistent impact of early parental deprivation on gene expression throughout their lives.
Upon analyzing the gene expression data, we identified 613 genes that exhibited significant differential expression in the hippocampus of human hand-reared young marmosets and 567 genes in human hand-reared aged marmosets, when compared to parent-reared age-matched marmosets.To explore the overlap between these two developmental stages, we performed a Venn diagram analysis.
The Venn diagram analysis revealed that among the differentially expressed genes, only 24 genes were upregulated (Fig. 4a, Table 2) and 4 genes were down-regulated in both young and aged stages (Fig. 4b, Table 3).This suggests that there is a limited overlap in the gene expression changes associated with early parental deprivation across the two developmental stages.
To further validate our Microarray analysis findings, we conducted real-time PCR analysis to assess the expression levels of KRT1, LCN2, OMD, and SLPI genes in the hippocampus, which were found to be downregulated in both young and aged stages (Fig. 4b, Table 3).The results of the real-time PCR analysis confirmed that the expression levels of these genes were consistently affected by early parental deprivation, which was consistent with the findings obtained from the microarray analysis (Fig. 4c).In addition to the gene expression analysis, we also conducted a DNA methylation analysis of the promoter region of the LCN2 gene.This analysis was performed to investigate the potential involvement of DNA methylation, specifically in the promoter region of LCN2, which is known to be one of the target genes of MECP2 38 .MECP2 indeed functions as a transcription factor capable of binding to methylated CpG sites, thereby influencing the regulation of downstream gene expression, either positively or negatively.By examining the DNA methylation patterns in the promoter region of LCN2 using bisulfite sequencing, we aimed to explore the potential epigenetic mechanisms underlying the continuous alterations in gene expression observed in response to early parental deprivation.However, the results of the DNA Table 1.Gene ontology (GO) enrichment analysis toward the 425 genes that were upregulated in human hand-reared marmosets.methylation analysis did not provide insights into the regulatory mechanisms involved in the persistent changes in LCN2 gene expression and their association with early life experiences and parental deprivation (Fig. 4d).We did not observe any significant differences in DNA methylation levels in the hippocampus of human hand-reared young and aged marmosets, in comparison to parent-reared age-matched marmosets.It is important to note that while DNA methylation is a well-known epigenetic mechanism, it is not the sole determinant of gene expression regulation.Other epigenetic modifications, such as histone modifications or non-coding RNA molecules, could be involved in mediating the effects of early parental deprivation on gene expression.Further investigations into alternative epigenetic mechanisms and regulatory processes may provide a more comprehensive understanding of the underlying mechanisms involved in the observed alterations in gene expression.

Discussion
This study represents the first investigation into the effects of early parental deprivation on gene expression changes in in common marmosets at two developmental stages, Young and Aged, using microarray analysis.
While previous research has predominantly concentrated on exploring the effects of maternal separation in rodent models 9,10,24,25 , it is crucial to note that the differences in the central stress response system between rodents and primates do not fully capture the complex dynamics of parent loss, abuse, or neglect observed in human populations 12,13 .Furthermore, studies conducted with rodents or non-human primates have demonstrated that repeated maternal separation, typically occurring for several hours per day during the initial or second week of infancy, can trigger acute stress responses 9,10,[14][15][16][17]24,25 . Convesely, non-human primates separated from their parents shortly after birth tend to exhibit a less pronounced acute stress response 21 .Interestingly, this study observed no significant differences in body weight between human hand-reared and parent-reared marmosets from birth (Fig. 1c).However, marmosets experiencing repeated parental deprivation in infancy displayed reduced body weight and elevated levels of ACTH and cortisol compared to the control group as previously described [14][15][16] .These findings highlight variations in the maternal separation paradigm and emphasize the importance of considering these differences when investigating the impact of early life experiences on stress physiology and behavior.In the present study, we observed upregulation of genes belonging to the Neuroactive-Ligand Receptor Interaction Pathway, including the OXTR gene, in young marmosets that underwent human hand-rearing. Prvious experiments conducted on rhesus macaques have reported a decrease in the expression of the OXTR gene and changes in epigenetic modifications 21 , which contradicts the findings of the present study.However, it is important to note that the discrepancy in results may be attributed to the different developmental stages that were analyzed.In our marmoset experiments, similar to the rhesus macaques, we observed a slight   39 .Rats raised in social isolation have been shown to exhibit increased ethanol and cocaine intake, along with enhanced dopamine and VMAT2 levels, which may contribute to a heightened vulnerability to addiction 40 .This suggests that abnormal dopamine release in human hand-reared marmosets could be associated with an increased risk of addiction.
On the other hand, TAS2R5, another up-regulated gene, has been found to exhibit higher gene expression during manic states based on analysis of peripheral blood samples in bipolar disorder 41 .This gene is involved in taste perception and may play a role in the manifestation of manic symptoms.
In contrast, the three down-regulated genes targeted in this study were LCN2, SLPI, and OMD, all of which encode extracellular secretory proteins.Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-inflammatory and antibacterial properties, and it promotes wound healing 42 .Slpi null mutant mice show reduced regeneration of posterior column axons in response to sciatic nerve injury 43 , suggesting that its decreased expression in this study may contribute to abnormal behavior due to impaired stress response and neuronal inflammation rescue.Osteomodulin (OMD) is a gene localized to extracellular exosomes and is believed to regulate cell adhesion and bone calcification 44 .A study related to psychiatric disorders found decreased expression of the OMD gene in amygdala gene expression analysis of children born after exposure to valproic acid (VPA) during the 12th day of gestation 45 .The precise mechanism by which this occurs is not yet known, but it suggests that OMD may have an impact on brain function.LCN2 is a secreted protein involved in innate immunity and has been shown to regulate spine morphology and suppress neuronal excitability 46 .Lcn2 knockout (KO) mice exhibit anxiety and depression-like behavior, cognitive dysfunction, and changes in hippocampal brain cell structure 47 .Decreased expression of Lcn2 has also been observed in a mouse model of Rett syndrome 38 .Although the exact mechanism of reduced LCN2 expression is still unclear, these findings suggest that LCN2 may be involved in developmental disorders, such as Rett syndrome, and potentially plays a role in developmental abnormalities observed in human hand-reared marmosets.Overall, the analysis of differentially expressed genes in this study provides insights into the potential molecular mechanisms underlying the behavioral abnormalities observed in young marmosets subjected to human hand-rearing as described previously 19,20 .However, it is important to note that this study exclusively used male samples, potentially limiting the generalizability of the identified gene expression changes.To address this gap, future studies should explore whether similar alterations in gene expression occur in female samples, accounting for factors such as the menstrual cycle and other relevant variables.Furthermore, among the human hand-reared marmosets analyzed, some were offspring of human hand-reared mother marmosets, which raises concerns regarding potential effects (Fig. 1b).Despite this, any epigenetic mutations occurring in the parental generation are unlikely to be passed on to the offspring.Nevertheless, owing to the unverifiable breeding history of the grandparents in this study, definitive confirmation is not possible.If the grandparents were also human hand-reared, there remains a possibility that epigenetic mutations might have been transmitted to the individuals under examination in this study.The study highlights the long-term effects of the nurturing environment during infancy on gene expression.While stress is commonly associated with early life experiences, this particular study did not directly address stress as a variable.However, it is evident that the nurturing environment during infancy influenced gene expression in the hippocampus.Further investigations are needed to explore various aspects of the human hand-rearing paradigm, including the specific cellular targets affected by the nurturing environment.
In order to gain a comprehensive understanding of the human hand-reared marmoset brain, it will be essential to incorporate MRI imaging analysis 48 .This imaging technique can provide insights into the structural and functional characteristics of the brain, offering valuable information about how the nurturing environment impacts brain development.
Overall, further studies are warranted to elucidate the effects of the nurturing environment during infancy.By conducting comprehensive investigations, we can better understand the underlying mechanisms and implications of early life experiences on gene expression, stress physiology, and brain structure and function in human hand-reared marmosets.This knowledge may pave the way for exploring gene therapy as a potential intervention for individuals dealing with developmental disorders, an area of growing significance in research and healthcare.

Animals
All animal experiments were approved by the Institutional Animal Experimentation Committee of the Central Institute for Experimental Animals (CIEA: 20049A) and conducted in compliance with the CIEA Standard Guidelines.These guidelines align with the recommendations for the proper conduct of animal experiments established by the Science Council of Japan and were reported in accordance with the ARRIVE guidelines.
The marmosets used in this study were procured from CLEA Japan, a facility accredited by the Japanese Society for Laboratory Animals Resources (certification number: 22-041).All animals received appropriate care under the supervision of the Institutional Animal Care and Use Committee of CLEA Japan, Inc. until their transfer to the CIEA (Central Institute for Experimental Animals).Crucially, the dimensions of the housing enclosures (W550 mm, D390 mm, H700 mm) were meticulously designed to align with the guidelines set forth by the US National Research Council.This adherence underscores our commitment to providing an environment that caters to the animals' well-being.The animal room maintained a temperature range of 23-29 °C, humidity between 30 and 70%, and a 12-h light-dark cycle (lights on from 8:00 to 20:00).The marmosets had access to pellet-type CMS-1M feed (CLEA Japan Inc) and water ad libitum.Enrichment strategies were thoughtfully implemented, including the provision of wooden sticks and boards within the cages, affording the marmosets the opportunity to freely engage in climbing and resting behaviors.Notably, the enclosures were designed to facilitate visual contact and audible communication between marmosets housed in different cages.This study included eleven male marmosets, comprising six human hand-reared marmosets and five parentreared marmosets.Hand-rearing was performed for neonates that were excluded from parental care when three or four offspring were born.
Brain tissue sampling was performed when the marmosets were 13-19 months old as young marmosets and 88-164 months old as aged marmosets.

Hand-rearing
Hand-rearing was conducted at CLEA Japan Inc.When three or more neonates were born from the same litter, two remained with the mother, and the remaining individuals underwent human hand-rearing.Traditionally, we assigned larger, stronger offspring to parental care, while smaller or weaker ones were hand-reared at our facility.However, our recent findings demonstrate that parent marmosets effectively care for their young, regardless of size or vitality.Consequently, to enhance the survival rate of marmosets under human-hand rearing, we deliberately choose heavier and sturdier individuals for this care.Our hand-rearing protocol involved specific steps.Shortly after birth, the neonates were separated from their parents and individually placed in small plastic cages measuring 150 mm × 215 mm × 120 mm.These cages were located in a separate animal room, ensuring no visual or auditory contact with their parents, within a standard incubator.The incubator maintained a temperature range of 27-33 °C, humidity between 40 and 80%, and a 12-h light-dark cycle (lights on from 7:00 to 19:00).Each plastic cage was equipped with various surrogates such as soft diaper sheets that the neonate could wrap and cling to, a heating pad covered with artificial fur, to ensure the neonates' sense of security.
Despite individual housing from postnatal day 0 to 30, the setup facilitated visual and vocal contact among neonates in neighboring cages.At around 30 days of age, the neonates were transferred from the incubator to slightly larger cages measuring 210 mm × 270 mm × 300 mm, where three marmosets lived together.By 50 days of age, five animals resided in a mid-sized cage measuring 550 mm × 780 mm × 700 mm.
As the marmosets matured, the feeding frequency gradually decreased, allowing them to consume larger quantities of milk per feeding.The feeding schedule adapted as the marmosets matured.It started with four daily feedings (0.9 ml to 1.4 ml) from days 0 to 7, with an increase in formula concentration.From days 7 to 27, three feedings (1.6 ml to 5.6 ml) were supplemented with banana lining and vitamin D3, alongside a gradual increase in formula concentration.From days 28 to 59, milk volume increased (8.0 ml to 10 ml), and additional food was introduced twice daily.Post-60 days, each feeding comprised 12 ml of formula, accompanied by once-daily solid feed.Throughout these periods, human caregivers interacted with the marmosets solely during suckling times.
Care was provided by human caregivers until the marmosets reached three months of age, at which point they were successfully weaned.Following this milestone, two same-aged males were housed together in cages measuring 550 mm × 390 mm × 700 mm.
Our care approach strictly adhered to the guidelines outlined in the 'Common Marmoset Management Work Manual, ' developed under the supervision of the Institutional Animal Care and Use Committee of CLEA Japan Inc.

Brain sampling
Brain tissue sampling was performed as follows.The marmosets were first anesthetized with a mixture of 0.04 mg/ kg medetomidine, 0.4 mg/kg midazolam, 0.4 mg/kg butorphanol, and 0.4 mg/kg butanol.Deep anesthesia was induced by isoflurane inhalation (0.5-3%).To confirm complete loss of pain response, skin was gently pinched at several sites using tweezers.After confirming the absence of pain response, blood was drawn from the inferior vena cava, and the marmoset was euthanized.The skull was quickly removed, the hippocampus was dissected, and the brain was flash-frozen in liquid nitrogen.

Microarray analysis
Total RNA was extracted from eleven marmoset hippocampus tissues using the TRIzol Reagent with the PureLink RNA Mini Kit (Thermo Fisher Scientific, USA).RNA quality was assessed using the Agilent 2200 TapeStation (Agilent, USA).RNA samples with a RIN value greater than 5.7 and 28S/18S ratio greater than 0.9 were selected for subsequent experiments (Supplementary Table S2).The microarray analysis was performed following the manufacturer's protocol.Briefly, 0.2 µg of total RNA was used to prepare Cyanine-3 (Cy3)-labeled cRNA using the Low Input Quick Amp Labeling Kit (Agilent, USA), followed by RNAeasy column purification (Qiagen, USA).Dye incorporation and cRNA yield were measured using the NanoDrop ND-1000 Spectrophotometer.The fragmented Cy3-labeled cRNA was then hybridized to the Marmoset microarray 8x60K (G4858A#84626, Agilent, USA).After hybridization, the microarrays were washed and scanned using the Agilent DNA Microarray Scanner.The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent, USA) using default parameters, and the obtained data were normalized and filtered using three filters with GeneSpring software 14.9 (Agilent, USA).
The microarray data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE236481.

Figure 1 .
Figure 1.Overview of the experimental workflow & Monthly body weight measurements.(a) Overview of the experimental workflow.HRY hand-reared young marmoset, PRY parent-reared young marmoset, HRA handreared aged marmoset, PRA parent-reared aged marmoset.(b) Sample information.(c) Monthly body weight measurements of parent-reared marmosets and human hand-reared marmosets were taken for eleven months, starting from the day of their birth.

Figure 2 .
Figure 2.The pathway of neuroactive ligand-receptor interaction may play a significant role in the shortterm consequences of early parental deprivation.(a) A volcano plot was generated to visualize the results of the microarray analysis comparing human hand-reared young marmosets to parent-reared young marmosets.In the plot, 425 genes that exhibited upregulation in the human hand-reared marmosets were shown in red color (fold change > 2.0, P < 0.05).On the other hand, 188 genes that displayed downregulation were shown in blue color (fold change < −2.0, P < 0.05).(b) Gene Set Enrichment Analysis (GSEA) of the neuroactive-ligand receptor interaction pathway enriched in human hand-reared marmosets compared to parent-reared young marmosets.(c) Heat map was visualized neuroactive ligand-receptor interaction pathway genes.HRY handreared young marmoset, PRY parent-reared young marmoset, red higher expression, blue lower expression. https://doi.org/10.1038/s41598-023-51025-z

Figure 3 .
Figure 3. Identification of potential pathways and processes involved in the long-term consequences of early parental deprivation.(a) Volcano plot of human hand-reared aged marmosets vs parent-reared aged marmosets.In the plot, 381 genes that exhibited upregulation in the human hand-reared marmosets were shown in red color (fold change > 2.0, P < 0.05).On the other hand, 186 genes that displayed downregulation were shown in blue color (fold change < −2.0, P < 0.05).(b) GSEA of the BOQUEST STEM CELL UP, NABA ECM GLYCOPROTEINS, and WU CELL MIGRATION enriched in human hand-reared marmosets compared to parent-reared aged marmosets.(c) Heat Map illustrates the partially or completely overlapped genes among BOQUEST STEM CELL UP, NABA ECM GLYCOPROTEINS, and WU CELL MIGRATION gene sets.The intensity of the blue color indicates the degree of decreased expression.

Figure 4 .
Figure 4. Identification of genes continuously affected by early parental deprivation throughout life.(a) Venn diagram for detecting commonly upregulated genes in both human hand-reared young marmosets and aged marmosets.(b) Venn diagram for detecting commonly downregulated genes in both human hand-reared young marmosets and aged marmosets.(c) Real-time PCR analysis to validate the microarray results for expression levels of KRT1, LCN2, OMD, and SLPI genes in human hand-reared marmosets compare to parent-reared marmosets.These genes were found to be down-regulated in both young and aged stages.*p < 0.05, **P < 0.01 (d) DNA methylation analysis of the promoter region of the LCN2 gene.Genomic map of the Lcn2 gene locus; scale bar = 500 bp.The positions of primers used for pyrosequencing are indicated by arrowheads.HRY handreared young marmoset, PRY parent-reared young marmoset, HRA hand-reared aged marmoset, PRA parentreared aged marmoset. https://doi.org/10.1038/s41598-023-51025-zwww.nature.com/scientificreports/

Table 2 .
Commonly up-regulated genes list in both human hand-reared young marmosets and aged marmosets.

Table 3 .
Commonly down-regulated genes list in both human hand-reared young marmosets and aged marmosets.reduction in the expression of the OXTR gene during the Aged stage.These findings highlight the dynamic nature of gene expression in response to early life experiences and indicate that the effects of early parental deprivation on gene expression may vary across different stages of development.Additionally, we identified a set of genes that showed consistent alterations across developmental stages and were continuously affected by early parental deprivation.It is particularly interesting that some of these genes are involved in the development of neurodevelopmental disorders and neuronal function.The up-regulated gene SLC18A2(VMAT2) functions by packaging dopamine into vesicles in synaptic terminal neurons and releasing it during neurotransmission Vol:.(1234567890) Scientific Reports | (2024) 14:330 | https://doi.org/10.1038/s41598-023-51025-zwww.nature.com/scientificreports/