Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

Low-frequency stimulation enhances ensemble co-firing and dexterity after stroke.

Electrical stimulation is a promising tool for modulating brain networks. However, it is unclear how stimulation interacts with neural patterns underlying behavior. Specifically, how might external stimulation that is not sensitive to the state of ongoing neural dynamics reliably augment neural processing and improve function? Here, we tested how low-frequency epidural alternating current stimulation (ACS) in non-human primates recovering from stroke interacted with task-related activity in perilesional cortex and affected grasping. We found that ACS increased co-firing within task-related ensembles and improved dexterity. Using a neural network model, we found that simulated ACS drove ensemble co-firing and enhanced propagation of neural activity through parts of the network with impaired connectivity, suggesting a mechanism to link increased co-firing to enhanced dexterity. Together, our results demonstrate that ACS restores neural processing in impaired networks and improves dexterity following stroke. More broadly, these results demonstrate approaches to optimize stimulation to target neural dynamics.

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

CD4+ T cells re-wire granuloma cellularity and regulatory networks to promote immunomodulation following Mtb reinfection.

Immunological priming-in the context of either prior infection or vaccination-elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrated that prior Mtb infection elicited a long-lasting protective response against subsequent Mtb exposure and was CD4+ T cell dependent. By analyzing data from primary infection, reinfection, and reinfection-CD4+ T cell-depleted granulomas, we found that the presence of CD4+ T cells during reinfection resulted in a less inflammatory lung milieu characterized by reprogrammed CD8+ T cells, reduced neutrophilia, and blunted type 1 immune signaling among myeloid cells. These results open avenues for developing vaccines and therapeutics that not only target lymphocytes but also modulate innate immune cells to limit tuberculosis (TB) disease.

Fetal Neuropathology in Zika Virus-Infected Pregnant Female Rhesus Monkeys.

The development of interventions to prevent congenital Zika syndrome (CZS) has been limited by the lack of an established nonhuman primate model. Here we show that infection of female rhesus monkeys early in pregnancy with Zika virus (ZIKV) recapitulates many features of CZS in humans. We infected 9 pregnant monkeys with ZIKV, 6 early in pregnancy (weeks 6-7 of gestation) and 3 later in pregnancy (weeks 12-14 of gestation), and compared findings with uninfected controls. 100% (6 of 6) of monkeys infected early in pregnancy exhibited prolonged maternal viremia and fetal neuropathology, including fetal loss, smaller brain size, and histopathologic brain lesions, including microcalcifications, hemorrhage, necrosis, vasculitis, gliosis, and apoptosis of neuroprogenitor cells. High-resolution MRI demonstrated concordant lesions indicative of deep gray matter injury. We also observed spinal, ocular, and neuromuscular pathology. Our data show that vascular compromise and neuroprogenitor cell dysfunction are hallmarks of CZS pathogenesis, suggesting novel strategies to prevent and to treat this disease.

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

Medial to lateral frontal functional connectivity mapping reveals the organization of cingulate cortex.

The functional organization of the frontal lobe is a source of debate, focusing on broad functional subdivisions, large-scale networks, or local refined specificities. Multiple neurocognitive models have tried to explain how functional interactions between cingulate and lateral frontal regions contribute to decision making and cognitive control, but their neuroanatomical bases remain unclear. We provide a detailed description of the functional connectivity between cingulate and lateral frontal regions using resting-state functional MRI in rhesus macaques. The analysis focuses on the functional connectivity of the rostral part of the cingulate sulcus with the lateral frontal cortex. Data-driven and seed-based analysis revealed three clusters within the cingulate sulcus organized along the rostro-caudal axis: the anterior, mid, and posterior clusters display increased functional connectivity with, respectively, the anterior lateral prefrontal regions, face-eye lateral frontal motor cortical areas, and hand lateral frontal motor cortex. The location of these clusters can be predicted in individual subjects based on morphological landmarks. These results suggest that the anterior cluster corresponds to the anterior cingulate cortex, whereas the posterior clusters correspond to the face-eye and hand cingulate motor areas within the anterior midcingulate cortex. These data provide a comprehensive framework to identify cingulate subregions based on functional connectivity and local organization.

Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity.

Carrier-free naked mRNA vaccines may reduce the reactogenicity associated with delivery carriers; however, their effectiveness against infectious diseases has been suboptimal. To boost efficacy, we targeted the skin layer rich in antigen-presenting cells (APCs) and utilized a jet injector. The jet injection efficiently introduced naked mRNA into skin cells, including APCs in mice. Further analyses indicated that APCs, after taking up antigen mRNA in the skin, migrated to the lymph nodes (LNs) for antigen presentation. Additionally, the jet injection provoked localized lymphocyte infiltration in the skin, serving as a physical adjuvant for vaccination. Without a delivery carrier, our approach confined mRNA distribution to the injection site, preventing systemic mRNA leakage and associated systemic proinflammatory reactions. In mouse vaccination, the naked mRNA jet injection elicited robust antigen-specific antibody production over 6 months, along with germinal center formation in LNs and the induction of both CD4- and CD8-positive T cells. By targeting the SARS-CoV-2 spike protein, this approach provided protection against viral challenge. Furthermore, our approach generated neutralizing antibodies against SARS-CoV-2 in non-human primates at levels comparable to those observed in mice. In conclusion, our approach offers a safe and effective option for mRNA vaccines targeting infectious diseases.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

Loss of TDP-43 mediates severe neurotoxicity by suppressing PJA1 gene transcription in the monkey brain.

The nuclear loss and cytoplasmic accumulation of TDP-43 (TAR DNA/RNA binding protein 43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previously, we reported that the primate-specific cleavage of TDP-43 accounts for its cytoplasmic mislocalization in patients' brains. This prompted us to investigate further whether and how the loss of nuclear TDP-43 mediates neuropathology in primate brain. In this study, we report that TDP-43 knockdown at the similar effectiveness, induces more damage to neuronal cells in the monkey brain than rodent mouse. Importantly, the loss of TDP-43 suppresses the E3 ubiquitin ligase PJA1 expression in the monkey brain at transcriptional level, but yields an opposite upregulation of PJA1 in the mouse brain. This distinct effect is due to the species-dependent binding of nuclear TDP-43 to the unique promoter sequences of the PJA1 genes. Further analyses reveal that the reduction of PJA1 accelerates neurotoxicity, whereas overexpressing PJA1 diminishes neuronal cell death by the TDP-43 knockdown in vivo. Our findings not only uncover a novel primate-specific neurotoxic contribution to the loss of function theory of TDP-43 proteinopathy, but also underscore a potential therapeutic approach of PJA1 to the loss of nuclear TDP-43.

A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Efficacy of Doxycycline and Ciprofloxacin for Treatment of Pneumonic Tularemia in Cynomolgus Macaques.

BACKGROUND: The incidence of pneumonic tularemia is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCMs) in humans. The US Food and Drug Administration's Animal Model Qualification Program under the Drug Development Tools Program is a regulatory pathway for animal models used in MCM efficacy testing and approval under the Animal Rule. The National Institute of Allergy and Infectious Diseases and Biomedical Advanced Research and Development Authority worked together to qualify the cynomolgus macaque model of pneumonic tularemia. METHODS: Using the model parameters and end points defined in the qualified model, efficacy of the antibiotics doxycycline and ciprofloxacin was evaluated in separate studies. Antibiotic administration, aimed to model approved human dosing, was initiated at time points of 24 hours or 48 hours after onset of fever as an indicator of disease. RESULTS: Upon aerosol exposure (target dose of 1000 colony-forming units) to Francisella tularensis SchuS4, 80% of vehicle-treated macaques succumbed or were euthanized. Ciprofloxacin treatment led to 10 of 10 animals surviving irrespective of treatment time. Doxycycline administered at 48 hours post-fever led to 10 of 10 animals surviving, while 9/10 animals survived in the group treated with doxycycline 24 hours after fever. Selected surviving animals in both the placebo and doxycycline 48-hour group showed residual live bacteria in peripheral tissues, while there were no bacteria in tissues from ciprofloxacin-treated macaques. CONCLUSIONS: Both doxycycline and ciprofloxacin were efficacious in treatment of pneumonic tularemia, although clearance of bacteria may be different between the 2 drugs.

Comparing the gut microbiota of Sichuan golden monkeys across multiple captive and wild settings: roles of anthropogenic activities and host factors.

BACKGROUND: Captivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision. RESULTS: Both captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota. CONCLUSIONS: Our aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.

Assessing cerebrovascular reactivity (CVR) in rhesus macaques (Macaca mulatta) using a hypercapnic challenge and pseudo-continuous arterial spin labeling (pCASL).

Cerebrovascular reactivity (CVR) is a measure of cerebral small vessels' ability to respond to changes in metabolic demand and can be quantified using magnetic resonance imaging (MRI) coupled with a vasoactive stimulus. Reduced CVR occurs with neurodegeneration and is associated with cognitive decline. While commonly measured in humans, few studies have evaluated CVR in animal models. Herein, we describe methods to induce hypercapnia in rhesus macaques (Macaca mulatta) under gas anesthesia to measure cerebral blood flow (CBF) and CVR using pseudo-continuous arterial spin labeling (pCASL). Fifteen (13 M, 2 F) adult rhesus macaques underwent pCASL imaging that included a baseline segment (100% O2) followed by a hypercapnic challenge (isoflurane anesthesia with 5% CO2, 95% O2 mixed gas). Relative hypercapnia was defined as an end-tidal CO2 (ETCO2) ≥5 mmHg above baseline ETCO2. The mean ETCO2 during the baseline segment of the pCASL sequence was 34 mmHg (range: 23-48 mmHg). During this segment, mean whole-brain CBF was 51.48 ml/100g/min (range: 21.47-77.23 ml/100g/min). Significant increases (p<0.0001) in ETCO2 were seen upon inspiration of the mixed gas (5% CO2, 95% O2). The mean increase in ETCO2 was 8.5 mmHg and corresponded with a mean increase in CBF of 37.1% (p<0.0001). The mean CVR measured was 4.3%/mmHg. No anesthetic complications occurred as a result of the CO2 challenge. Our methods were effective at inducing a state of relative hypercapnia that corresponds with a detectable increase in whole brain CBF using pCASL MRI. Using these methods, a CO2 challenge can be performed in conjunction with pCASL imaging to evaluate CBF and CVR in rhesus macaques. The measured CVR in rhesus macaques is comparable to human CVR highlighting the translational utility of rhesus macaques in neuroscience research. These methods present a feasible means to measure CVR in comparative models of neurodegeneration and cerebrovascular dysfunction.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

Spontaneous playful teasing in four great ape species.

Joking draws on complex cognitive abilities: understanding social norms, theory of mind, anticipating others' responses and appreciating the violation of others' expectations. Playful teasing, which is present in preverbal infants, shares many of these cognitive features. There is some evidence that great apes can tease in structurally similar ways, but no systematic study exists. We developed a coding system to identify playful teasing and applied it to video of zoo-housed great apes. All four species engaged in intentionally provocative behaviour, frequently accompanied by characteristics of play. We found playful teasing to be characterized by attention-getting, one-sidedness, response looking, repetition and elaboration/escalation. It takes place mainly in relaxed contexts, has a wide variety of forms, and differs from play in several ways (e.g. asymmetry, low rates of play signals like the playface and absence of movement-final 'holds' characteristic of intentional gestures). As playful teasing is present in all extant great ape genera, it is likely that the cognitive prerequisites for joking evolved in the hominoid lineage at least 13 million years ago.

Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.