Human-like adrenal features in Chinese tree shrews revealed by multi-omics analysis of adrenal cell populations and steroid synthesis

The Chinese tree shrew(Tupaia belangeri chinensis)has emerged as a promising model for investigating adrenal steroid synthesis,but it is unclear whether the same cells produce steroid hormones and whether their production is regulated in the same way as in humans.Here,we comprehensively mapped the cell types and pathways of steroid metabolism in the adrenal gland of Chinese tree shrews using single-cell RNA sequencing,spatial transcriptome analysis,mass spectrometry,and immunohistochemistry.We compared the transcriptomes of various adrenal cell types across tree shrews,humans,macaques,and mice.Results showed that tree shrew adrenal glands expressed many of the same key enzymes for steroid synthesis as humans,including CYP11B2,CYP11B1,CYB5A,and CHGA.Biochemical analysis confirmed the production of aldosterone,cortisol,and dehydroepiandrosterone but not dehydroepiandrosterone sulfate in the tree shrew adrenal glands.Furthermore,genes in adrenal cell types in tree shrews were correlated with genetic risk factors for polycystic ovary syndrome,primary aldosteronism,hypertension,and related disorders in humans based on genome-wide association studies.Overall,this study suggests that the adrenal glands of Chinese tree shrews may consist of closely related cell populations with functional similarity to those of the human adrenal gland.Our comprehensive results(publicly available at http://gxmujyzmolab.cn:16245/scAGMap/)should facilitate the advancement of this animal model for the investigation of adrenal gland disorders.

Identification of novel mammalian viruses in tree shrews(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis),a member of the mammalian order Scandentia,exhibits considerable similarities with primates,including humans,in aspects of its nervous,immune,and metabolic systems.These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer,infectious diseases,metabolic disorders,and mental health conditions.Herein,we used metatranscriptomic sequencing to analyze plasma,as well as oral and anal swab samples,from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses.In total,eight mammalian viruses with complete genomes were identified,belonging to six viral families,including Flaviviridae,Hepeviridae,Parvovirinae,Picornaviridae,Sedoreoviridae,and Spinareoviridae.Notably,the presence of rotavirus was recorded in tree shrews for the first time.Three viruses-hepacivirus 1,parvovirus,and picornavirus-exhibited low genetic similarity(<70%)with previously reported viruses at the whole-genome scale,indicating novelty.Conversely,three other viruses-hepacivirus 2,hepatovirus A and hepevirus-exhibited high similarity(>94%)to known viral strains.Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants.These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews,highlighting the necessity for further research into their potential for crossspecies transmission.

Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells

Tree shrews(Tupaia belangeri chinensis)share a close relationship to primates and have been widely used in biomedical research.We previously established a spermatogonial stem cell(SSC)-based gene editing platform to generate transgenic tree shrews.However,the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear.Here,we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages.We found that SSCs lost spermatogenesis ability after long-term expansion(>50 passages),as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia(SPG)-derivedspermatocytesor spermatids marking spermatogenesis.RNA sequencing(RNA-seq)analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers.Specifically,DNA damage response and repair genes(e.g.,MRE11,SMC3,BLM,and GEN1)were down-regulated,whereas genes associated with mitochondrial function(e.g.,NDUFA9,NDUFA8,NDUFA13,and NDUFB8)were up-regulated after expansion.The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells.Supplementation with nicotinamide adenine dinucleotide(NAD+)precursor nicotinamide riboside(NR)exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture.Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.

清醒活动树鼩Tupaia(Tree Shrew)神经元单位放电的记录

鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报道,本文报告一种记录清醒活动状态树鼩的单位放电技术考虑到树鼩形体甚小、体重仅100多克。要分离单位放电,必须拥有一种特殊规格的微型微推进器,以适应特殊实验之需。本研究设计一种采用差动结构原理实现微推进的装置,整个装置由微推进器与基座组成。微推进器的参数如下:重量8.8克;微调范围:2.0毫米;微调读数5微米;微调可控范围:小于2微米;外形尺寸:13×13×51毫米。微推进器相对于基座有直径为1.8毫米的径向偏心移动范围。基座重0.65克,实验在局麻下,在立体定向仪控制下埋植基座。术后第二天即可实验观察。

Creating animal models, why not use the Chinese tree shrew ( Tupaia belangeri chinensis)？

The Chinese tree shrew （Tupaia belangeri chinensis） a squirrel-like and rat-sized mammal, has a wide distribution in Southeast Asia, South and Southwest China and has many unique characteristics that make it suitable for use as an experimental animal. There have been many studies using the tree shrew （Tupaia belangeri） aimed at increasing our understanding of fundamental biological mechanisms and for the modeling of human diseases and therapeutic responses. The recent release of a publicly available annotated genome sequence of the Chinese tree shrew and its genome database （www.treeshrewdb.org） has offered a solid base from which it is possible to elucidate the basic biological properties and create animal models using this species. The extensive characterization of key factors and signaling pathways in the immune and nervous systems has shown that tree shrews possess both conserved and unique features relative to primates. Hitherto, the tree shrew has been successfully used to create animal models for myopia, depression, breast cancer, alcohol-induced or non-alcoholic fatty liver diseases, herpes simplex virus type 1 （HSV-1） and hepatitis C virus （HCV） infections, to name a few. The recent successful genetic manipulation of the tree shrew has opened a new avenue for the wider usage of this animal in biomedical research. In this opinion paper, I attempt to summarize the recent research advances that have used the Chinese tree shrew, with a focus on the new knowledge obtained by using the biological properties identified using the tree shrew genome, a proposal for the genome-based approach for creating animal models, and the genetic manipulation of the tree shrew. With more studies using this species and the application of cutting-edge gene editing techniques, the tree shrew will continue to be under the spot light as a viable animal model for investigating the basis of many different human diseases.

Tree shrew (Tupaia belangeri)as a novel laboratory disease animal model

The tree shrew （Tupaia belangeri） is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.

Comprehensive annotation of the Chinese tree shrew genome by large-scale RNA sequencing and long-read isoform sequencing

The Chinese tree shrew(Tupaia belangeri chinensis)is emerging as an important experimental animal in multiple fields of biomedical research.Comprehensive reference genome annotation for both mRNA and long non-coding RNA(lncRNA)is crucial for developing animal models using this species.In the current study,we collected a total of 234 high-quality RNA sequencing(RNA-seq)datasets and two long-read isoform sequencing(ISO-seq)datasets and improved the annotation of our previously assembled high-quality chromosomelevel tree shrew genome.We obtained a total of 3514 newly annotated coding genes and 50576 lncRNA genes.We also characterized the tissuespecific expression patterns and alternative splicing patterns of mRNAs and lncRNAs and mapped the orthologous relationships among 11 mammalian species using the current annotated genome.We identified 144 tree shrew-specific gene families,including interleukin 6(IL6)and STT3 oligosaccharyltransferase complex catalytic subunit B(STT3B),which underwent significant changes in size.Comparison of the overall expression patterns in tissues and pathways across four species(human,rhesus monkey,tree shrew,and mouse)indicated that tree shrews are more similar to primates than to mice at the tissue-transcriptome level.Notably,the newly annotated purine rich element binding protein A(PURA)gene and the STT3B gene family showed dysregulation upon viral infection.The updated version of the tree shrew genome annotation(KIZ version 3:TS_3.0)is available at http://www.treeshrewdb.org and provides an essential reference for basic and biomedical studies using tree shrew animal models.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5- hydroxymethylcytosine （5hmC） ten-eleven transtocation （TET） enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews （Tupaia belangeri chinensis）. The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis)is a small experimental animal with a close affinity to primates.This species has long been proposed to be an alternative experimental animal to primates in biomedical research.Despite decades of study,there is no pure breed for this animal,and the overall genetic diversity of wild tree shrews remains largely unknown.In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations,we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew.An analysis of a wild population of 117 individuals collected from the suburb of Kunming,China,showed that these loci exhibited a highly expected heterozygosity(0.616).These 12 microsatellites were sufficient for individual identification and parentage analysis.The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.

A bright future for the tree shrew in neuroscience research:Summary from the inaugural Tree Shrew Users Meeting

Tree shrews(Tupaia spp.)have been used in neuroscience research since the 1960s due to their evolutionary proximity to primates.The use of and interest in this animal model have recently increased,in part due to the adaptation of modern neuroscience tools in this species.These tools include quantitative behavioral assays,calcium imaging,optogenetics and transgenics.To facilitate the exchange and development of these new technologies and associated research findings,we organized the inaugural“Tree Shrew Users Meeting”which was held online due to the COVID-19 pandemic.Here,we review this meeting and discuss the history of tree shrews as an animal model in neuroscience research and summarize the current themes being investigated using this animal,as well as future directions.

Basal physiological parameters in domesticated tree shrews(Tupaia belangeri chinensis)

Establishing non-human primate models of human diseases is an efficient way to narrow the large gap between basic studies and translational medicine.Multifold advantages such as simplicity of breeding,low cost of feeding and facility of operating make the tree shrew an ideal non-human primate model proxy.Additional features like vulnerability to stress and spontaneous diabetic characteristics also indicate that the tree shrew could be a potential new animal model of human diseases.However,basal physiological indexes of tree shrew,especially those related to human disease,have not been systematically reported.Accordingly,we established important basal physiological indexes of domesticated tree shrews including several factors:(1)body weight,(2)core body temperature and rhythm,(3)diet metabolism,(4)locomotor rhythm,(5)electroencephalogram,(6)glycometabolism and(7)serum and urinary hormone level and urinary cortisol rhythm.We compared the physiological parameters of domesticated tree shrew with that of rats and macaques.Results showed that(a)the core body temperature of the tree shrew was 39.59±0.05°C,which was higher than that of rats and macaques;(b)Compared with wild tree shrews,with two activity peaks,domesticated tree shrews had only one activity peak from 17:30 to 19:30;(c)Compared with rats,tree shrews had poor carbohydrate metabolism ability;and(d)Urinary cortisol rhythm indicated there were two peaks at 8:00 and 17:00 in domesticated tree shrews,which matched activity peaks in wild tree shrews.These results provided basal physiological indexes for domesticated tree shrews and laid an important foundation for diabetes and stress-related disease models established on tree shrews.

Early embryonic development and transplantation in tree shrews

As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained （conception rate=74%）. Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor（100 μg/L） to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

Alteration of the p53 gene during tree shrews' hepatocarcinogenesis

OBJECTIVE: To detect the expression and variation of the p53 gene in hepatocarcinogenesis of tree shrews induced by hepatitis B virus (HBV) and aflatoxin B_1(AFB_1). METHODS: Tree shrews were divided into four groups: group A, infected with HBV and fed with AFB_1; group B, only infected with HBV; group C, fed with AFB_1 alone; and group D normal control. The tree shrews underwent liver biopsy every. 15 weeks. Liver and tumor tissues were detected by immunohistochemistry and molecular biotechnologies. RESULTS: The incidence of hepatocellular carcinoma (HCC) was higher in group A (66.7%) than in groups B (0) and C (30%). HCC occurrence was earlier in group A than in group C (120.0±16.6 wk vs 153.3±5.8 wk, t=3.336, P<0.01). Mutated p53 protein was not found in all groups before 75 weeks of experiment. At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60.0% and 71.4% in groups A, B and C respectively, which were significantly higher than that in group D (10%) (X^2≥5.03, P<0.05). An abnormal band of the p53 gene was detected in groups A and C. The mutational points of the p53 gene in liver cancer of tree shrews were at codon 275, 78 and 13. Nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 were 91.7% and 93.4% in homology, compared with those of human p53, respectively. CONCLUSIONS: Remarkable synergistic effect on HCC exists between HBV and AFB_1. Mutated p53 protein expressed before occurrence of HCC promotes the development of HCC. HBV and AFB_1 may synergistically induce p53 gene mutation.

Age-related changes of corneal endothelial cell in healthy Chinese tree shrew measured by non-contact specular microscope

AIM：To determine the impact of age on the morphology of endothelial cells and central corneal thickness（CCT） in Chinese tree shrew.METHODS：One-hundred and twenty eyes of 60 healthy Chinese tree shrews were studied.Based on age,the tree shrews were divided into four groups.After general anesthesia,the images of endothelium were acquired using non-contact specular microscope Topcon 3000 P.Eight parameters of corneal endothelial cells were measured by built-in software,including CCT,endothelial cell density（ECD）,percent hexagonality（HG%）,coeffi cient of variability（CV）,size of minimal cell（Smin）,size of maximal cell（Smax）,average cells size（Savg） and size standard deviation（Ssd）.Data were analyzed using STATA software.The differences of eight parameters among groups and correlations with age were analyzed.RESULTS：In all studied animals,the average CCT was 2 4 9.6 ± 2 .29 μ m（202-301 μm）,ECD was 308 0.72 ± 460.76 cells/mm^2（1239.6-4047.6 cells/mm^2） and CV was 29.10±7.60（13.6-54.6）.CV was significantly different among different groups（P〈0.001）.Strong correlation with age was found in ECD,Smax,Savg,Ssd and CV.CONCLUSION：Cornea of Chinese tree shrews had half CCT of human cornea and similar ECD,CV and size of corneal endothelial cells.Young adult tree shrews had higher ECD,HG% and low CV.ECD,Smax,Savg,Ssd and CV correlated with age signifi cantly.

EXPERIMENTAL PRIMARY LIVER CANCER IN TREE SHREWS EXPOSED TO HUMAN HEPATITIS B VIRUS AND AFLATOXIN B_(1)

On the basis of the successful establishment of an animal model in tree shrews experimentally in fected with human hepatitis B virus (HHBV), a study on the hepatocarcinogenic effects of HHBV and aflatoxin B1 (AFB1) by using this animal model was conducted through a lifelong experiment. Among 41 tree shrews exposed to AFB1, 17 were experimentally infected by HHBV and 24 were uninfected. After 158 weeks, significant difference of primary liver cancer (PLC) incidence was present between the HHBV infected (52.94%) and uninfected (12.5%) groups (p<0.05). No difference was found between these two groups in the amount of AFB4 ingestion. Moreover, 1/9 of the tree shrews infected only by HHBV but not exposed to AFB4 developed PLC. No PLC was found in 6 tree shrews that had neither been infected with HHBV nor been exposed to AFB4. These results suggest the possible etiologic relationship between HHBV infection and PLC, as well as the synergetic effects of HHBV and AFB4 during PLC development.

Experimental infection of tree shrews(Tupaia belangeri) with Coxsackie virus A16

Coxsackie virus A16(CA16) is commonly recognized as one of the main human pathogens of hand-foot-mouth disease(HFMD). The clinical manifestations of HFMD include vesicles of hand, foot and mouth in young children and severe inflammatory CNS lesions. In this study, experimentally CA16 infected tree shrews(Tupaia belangeri) were used to investigate CA16 pathogenesis. The results showed that both the body temperature and the percentages of blood neutrophilic granulocytes / monocytes of CA16 infected tree shrews increased at 4-7 days post infection. Dynamic distributions of CA16 in different tissues and stools were found at different infection stages. Moreover, the pathological changes in CNS and other organs were also observed. These findings indicate that tree shrews can be used as a viable animal model to study CA16 infection.

Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews

Endosymbionts influence many aspects of their hosts’ health conditions, including physiology, development, immunity, metabolism, etc. Tree shrews(Tupaia belangeri chinensis) have attracted increasing attention in modeling human diseases and therapeutic responses due to their close relationship with primates. To clarify the situation of symbiotic bacteria from their body surface, oral cavity, and anus, 12 wild and 12 the third generation of captive tree shrews were examined. Based on morphological and cultural characteristics, physiological and biochemical tests, as well as the 16 S rDNA full sequence analysis, 12 bacteria strains were isolated and identified from the wild tree shrews: body surface: Bacillus subtilis(detection rate 42%), Pseudomonas aeruginosa(25%), Staphlococcus aureus(33%), S. Epidermidis(75%), Micrococcus luteus(25%), Kurthia gibsonii(17%); oral cavity: Neisseria mucosa(58%), Streptococcus pneumonia(17%); anus: Enterococcus faecalis(17%), Lactococus lactis(33%), Escherichia coli(92%), Salmonella typhosa(17%); whereas, four were indentified from the third generation captive tree shrews: body surface: S. epidermidis(75%); oral cavity: N.mucosa(67%); anus: L. lactis(33%), E. coli(100%). These results indicate that S. epidermidis, N. mucosa, L. lactis and E. coli were major bacteria in tree shrews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa were species-specific flora. This study facilitates the future use of tree shrews as a standard experimental animal and improves our understanding of the relationship between endosymbionts and their hosts.

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew (Tupaia belangeri chinensis)

The Chinese tree shrew (Tupaia belangeri chinensis) is a small experimental animal with a close affinity to primates. This species has long been proposed to be an alternative experimental animal to primates in biomedical research. Despite decades of study, there is no pure breed for this animal, and the overall genetic diversity of wild tree shrews remains largely unknown. In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations, we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew. An analysis of a wild population of 117 individuals collected from the suburb of Kunming, China, showed that these loci exhibited a highly expected heterozygosity (0.616). These 12 microsatellites were sufficient for individual identification and parentage analysis. The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.

Basal physiological parameters in domesticated tree shrews (Tupaia belangeri chinensis)

Establishing non-human primate models of human diseases is an efficient way to narrow the large gap between basic studies and translational medicine. Multifold advantages such as simplicity of breeding, low cost of feeding and facility of operating make the tree shrew an ideal non-human primate model proxy. Additional features like vulnerability to stress and spontaneous diabetic characteristics also indicate that the tree shrew could be a potential new animal model of human diseases. However, basal physiological indexes of tree shrew, especially those related to human disease, have not been systematically reported. Accordingly, we established important basal physiological indexes of domesticated tree shrews including several factors: (1) body weight, (2) core body temperature and rhythm, (3) diet metabolism, (4) locomotor rhythm, (5) electroencephalogram, (6) glycometabolism and (7) serum and urinary hormone level and urinary cortisol rhythm. We compared the physiological parameters of domesticated tree shrew with that of rats and macaques. Results showed that (a) the core body temperature of the tree shrew was 39.59±0.05 °C, which was higher than that of rats and macaques; (b) Compared with wild tree shrews, with two activity peaks, domesticated tree shrews had only one activity peak from 17:30 to 19:30; (c) Compared with rats, tree shrews had poor carbohydrate metabolism ability; and (d) Urinary cortisol rhythm indicated there were two peaks at 8:00 and 17:00 in domesticated tree shrews, which matched activity peaks in wild tree shrews. These results provided basal physiological indexes for domesticated tree shrews and laid an important foundation for diabetes and stress-related disease models established on tree shrews.