2023 Vol. 44, No. 3

2023-3 Contents 
2023, 44(3)
Chronic liver injury leads to progressive liver fibrosis and ultimately cirrhosis, a major cause of morbidity and mortality worldwide. However, there are currently no effective anti-fibrotic therapies available, especially for late-stage patients, which is partly attributed to the major knowledge gap regarding liver cell heterogeneity and cell-specific responses in different fibrosis stages. To reveal the multicellular networks regulating mammalian liver fibrosis from mild to severe phenotypes, we generated a single-nucleus transcriptomic atlas encompassing 49 919 nuclei corresponding to all main liver cell types at different stages of murine carbon tetrachloride (CCl4)-induced progressive liver fibrosis. Integrative analysis distinguished the sequential responses to injury of hepatocytes, hepatic stellate cells and endothelial cells. Moreover, we reconstructed the cell-cell interactions and gene regulatory networks implicated in these processes. These integrative analyses uncovered previously overlooked aspects of hepatocyte proliferation exhaustion and disrupted pericentral metabolic functions, dysfunction for clearance by apoptosis of activated hepatic stellate cells, accumulation of pro-fibrotic signals, and the switch from an anti-angiogenic to a pro-angiogenic program during CCl4-induced progressive liver fibrosis. Our dataset thus constitutes a useful resource for understanding the molecular basis of progressive liver fibrosis using a relevant animal model.
Chalcidoidea is one of the most biologically diverse groups among Hymenoptera. Members are characterized by extraordinary parasitic lifestyles and extensive host ranges, among which several species attack plants or serve as pollinators. However, higher-level chalcidoid relationships remain controversial. Here, we performed mitochondrial phylogenomic analyses for major clades (18 out of 25 families) of Chalcidoidea based on 139 mitochondrial genomes. The compositional heterogeneity and conflicting backbone relationships in Chalcidoidea were assessed using various datasets and tree inferences. Our phylogenetic results supported the monophyly of 16 families and polyphyly of Aphelinidae and Pteromalidae. Our preferred topology recovered the relationship (Mymaridae+(Signiphoridae+Leucospidae)+(Chalcididae+((Perilampidae+Eucharitidae)+ remaining Chalcidoidea)))). The monophyly of Agaonidae and Sycophaginae was rejected, while the gall-associated ((Megastigmidae+Ormyridae)+(Ormocerinae+Eurytomidae)) relationship was supported in most results. A six-gene inversion may be a synapomorphy for most families, whereas other derived gene orders may introduce confusion in phylogenetic signals at deeper nodes. Dating estimates suggested that Chalcidoidea arose near the Jurassic/Cretaceous boundary and that two dynamic shifts in diversification occurred during the evolution of Chalcidoidea. We hypothesized that the potential codiversification between chalcidoids and their hosts may be crucial for accelerating the diversification of Chalcidoidea. Ancestral state reconstruction analyses supported the hypothesis that gall-inducers were mainly derived from parasitoids of gall-inducers, while other gall-inducers were derived from phytophagous groups. Taken together, these findings advance our understanding of mitochondrial genome evolution in the major interfamilial phylogeny of Chalcidoidea.
Despite the urgent need for conservation consideration, strategic action plans for the preservation of the Asian honeybee, Apis cerana Fabricius, 1793, remain lacking. Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy. Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts, as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities. Here, we investigated genome variations in 362 worker bees representing almost all populations of mainland A. cerana to understand how evolution has shaped its population structure. Whole-genome single nucleotide polymorphisms (SNPs) based on nuclear sequences revealed eight putative subspecies, with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies. Our results demonstrated that most classic morphological traits, including body size, were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism. Thus, such morphological traits were not suitable for subspecific delineation. Conversely, wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes. Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor. Based on our findings, we propose that criteria for subspecies delineation should be based on evolutionary independence, trait distinction, and geographic isolation. We formally defined and described eight subspecies of mainland A. cerana. Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units, guiding colony introduction and breeding.
The coronavirus disease 2019 (COVID-19) pandemic has greatly damaged human society, but the origins and early transmission patterns of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen remain unclear. Here, we reconstructed the transmission networks of SARS-CoV-2 during the first three and six months since its first report based on ancestor-offspring relationships using BANAL-52-referenced mutations. We explored the position (i.e., root, middle, or tip) of early detected samples in the evolutionary tree of SARS-CoV-2. In total, 6 799 transmission chains and 1 766 transmission networks were reconstructed, with chain lengths ranging from 1–9 nodes. The root node samples of the 1 766 transmission networks were from 58 countries or regions and showed no common ancestor, indicating the occurrence of many independent or parallel transmissions of SARS-CoV-2 when first detected (i.e., all samples were located at the tip position of the evolutionary tree). No root node sample was found in any sample (n=31, all from the Chinese mainland) collected in the first 15 days from 24 December 2019. Results using six-month data or RaTG13-referenced mutation data were similar. The reconstruction method was verified using a simulation approach. Our results suggest that SARS-CoV-2 may have already been spreading independently worldwide before the outbreak of COVID-19 in Wuhan, China. Thus, a comprehensive global survey of human and animal samples is essential to explore the origins of SARS-CoV-2 and its natural reservoirs and hosts.
Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1-Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.
Research highlight
Viral infections have led to many public health crises and pandemics in the last few centuries. Neurotropic virus infection-induced viral encephalitis (VE), especially the symptomatic inflammation of the meninges and brain parenchyma, has attracted growing attention due to its high mortality and disability rates. Understanding the infectious routes of neurotropic viruses and the mechanism underlying the host immune response is critical to reduce viral spread and improve antiviral therapy outcomes. In this review, we summarize the common categories of neurotropic viruses, viral transmission routes in the body, host immune responses, and experimental animal models used for VE study to gain a deeper understanding of recent progress in the pathogenic and immunological mechanisms under neurotropic viral infection. This review should provide valuable resources and perspectives on how to cope with pandemic infections.
Acute administration of MK-801 (dizocilpine), an N-methyl-D-aspartate receptor (NMDAR) antagonist, can establish animal models of psychiatric disorders. However, the roles of microglia and inflammation-related genes in these animal models of psychiatric disorders remain unknown. Here, we found rapid elimination of microglia in the prefrontal cortex (PFC) and hippocampus (HPC) of mice following administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor PLX3397 (pexidartinib) in drinking water. Single administration of MK-801 induced hyperactivity in the open-field test (OFT). Importantly, PLX3397-induced depletion of microglia prevented the hyperactivity and schizophrenia-like behaviors induced by MK-801. However, neither repopulation of microglia nor inhibition of microglial activation by minocycline affected MK-801-induced hyperactivity. Importantly, microglial density in the PFC and HPC was significantly correlated with behavioral changes. In addition, common and distinct glutamate-, GABA-, and inflammation-related gene (116 genes) expression patterns were observed in the brains of PLX3397- and/or MK-801-treated mice. Moreover, 10 common inflammation-related genes (CD68, CD163, CD206, TMEM119, CSF3R, CX3CR1, TREM2, CD11b, CSF1R, and F4/80) with very strong correlations were identified in the brain using hierarchical clustering analysis. Further correlation analysis demonstrated that the behavioral changes in the OFT were most significantly associated with the expression of inflammation-related genes (NLRP3, CD163, CD206, F4/80, TMEM119, and TMEM176a), but not glutamate- or GABA-related genes in PLX3397- and MK-801-treated mice. Thus, our results suggest that microglial depletion via a CSF1R/c-Kit kinase inhibitor can ameliorate the hyperactivity induced by an NMDAR antagonist, which is associated with modulation of immune-related genes in the brain.
KIZ-CUHK Joint Laboratory Special Column
Parkinson’s disease (PD) relates to defective mitochondrial quality control in the dopaminergic motor network. Genetic studies have revealed that PINK1 and Parkin mutations are indicative of a heightened propensity to PD onset, pinpointing mitophagy and inflammation as the culprit pathways involved in neuronal loss in the substantia nigra (SNpc). In a reciprocal manner, LRRK2 functions in the regulation of basal flux and inflammatory responses responsible for PINK1/Parkin-dependent mitophagy activation. Pharmacological intervention in these disease-modifying pathways may facilitate the development of novel PD therapeutics, despite the current lack of an established drug evaluation model. As such, we reviewed the feasibility of employing the versatile global Pink1 knockout (KO) rat model as a self-sufficient, spontaneous PD model for investigating both disease etiology and drug pharmacology. These rats retain clinical features encompassing basal mitophagic flux changes with PD progression. We demonstrate the versatility of this PD rat model based on the incorporation of additional experimental insults to recapitulate the proinflammatory responses observed in PD patients.
Congenital heart disease (CHD) is observed in up to 1% of live births and is one of the leading causes of mortality from birth defects. While hundreds of genes have been implicated in the genetic etiology of CHD, their role in CHD pathogenesis is still poorly understood. This is largely a reflection of the sporadic nature of CHD, as well as its variable expressivity and incomplete penetrance. We reviewed the monogenic causes and evidence for oligogenic etiology of CHD, as well as the role of de novo mutations, common variants, and genetic modifiers. For further mechanistic insight, we leveraged single-cell data across species to investigate the cellular expression characteristics of genes implicated in CHD in developing human and mouse embryonic hearts. Understanding the genetic etiology of CHD may enable the application of precision medicine and prenatal diagnosis, thereby facilitating early intervention to improve outcomes for patients with CHD.
Large animal models of cardiac ischemia-reperfusion are critical for evaluation of the efficacy of cardioprotective interventions prior to clinical translation. Nonetheless, current cardioprotective strategies/interventions formulated in preclinical cardiovascular research are often limited to small animal models, which are not transferable or reproducible in large animal models due to different factors such as: (i) complex and varied features of human ischemic cardiac disease (ICD), which are challenging to mimic in animal models, (ii) significant differences in surgical techniques applied, and (iii) differences in cardiovascular anatomy and physiology between small versus large animals. This article highlights the advantages and disadvantages of different large animal models of preclinical cardiac ischemic reperfusion injury (IRI), as well as the different methods used to induce and assess IRI, and the obstacles faced in using large animals for translational research in the settings of cardiac IR.
Generating diverse motor behaviors critical for survival is a challenge that confronts the central nervous system (CNS) of all animals. During movement execution, the CNS performs complex calculations to control a large number of neuromusculoskeletal elements. The theory of modular motor control proposes that spinal interneurons are organized in discrete modules that can be linearly combined to generate a variety of behavioral patterns. These modules have been previously represented as stimulus-evoked force fields (FFs) comprising isometric limb-endpoint forces across workspace locations. Here, we ask whether FFs elicited by different stimulations indeed represent the most elementary units of motor control or are themselves the combination of a limited number of even more fundamental motor modules. To probe for potentially more elementary modules, we optogenetically stimulated the lumbosacral spinal cord of intact and spinalized Thy1-ChR2 transgenic mice (n=21), eliciting FFs from as many single stimulation loci as possible (20–70 loci per mouse) at minimally necessary power. We found that the resulting varieties of FFs defied simple categorization with just a few clusters. We used gradient descent to further decompose the FFs into their underlying basic force fields (BFFs), whose linear combination explained FF variability. Across mice, we identified 4–5 BFFs with partially localizable but overlapping representations along the spinal cord. The BFFs were structured and topographically distributed in such a way that a rostral-to-caudal traveling wave of activity across the lumbosacral spinal cord may generate a swing-to-stance gait cycle. These BFFs may represent more rudimentary submodules that can be flexibly merged to produce a library of motor modules for building different motor behaviors.
Chemotherapy can significantly reduce follicle counts in ovarian tissues and damage ovarian stroma, causing endocrine disorder, reproductive dysfunction, and primary ovarian insufficiency (POI). Recent studies have suggested that extracellular vesicles (EVs) secreted from mesenchymal stem cells (MSCs) exert therapeutic effects in various degenerative diseases. In this study, transplantation of EVs from human induced pluripotent stem cell-derived MSCs (iPSC-MSC-EVs) resulted in significant restoration of ovarian follicle numbers, improved granulosa cell proliferation, and inhibition of apoptosis in chemotherapy-damaged granulosa cells, cultured ovaries, and in vivo ovaries in mice. Mechanistically, treatment with iPSC-MSC-EVs resulted in up-regulation of the integrin-linked kinase (ILK) -PI3K/AKT pathway, which is suppressed during chemotherapy, most likely through the transfer of regulatory microRNAs (miRNAs) targeting ILK pathway genes. This work provides a framework for the development of advanced therapeutics to ameliorate ovarian damage and POI in female chemotherapy patients.
Telomeres are nucleoprotein structures located at the end of each chromosome, which function in terminal protection and genomic stability. Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo. As relatively long-lived mammals based on body size, bats display unique telomeric patterns, including the up-regulation of genes involved in alternative lengthening of telomeres (ALT), DNA repair, and DNA replication. At present, however, the relevant molecular mechanisms remain unclear. In this study, we performed cross-species comparison and identified EPAS1, a well-defined oxygen response gene, as a key telomeric protector in bat fibroblasts. Bat fibroblasts showed high expression of EPAS1, which enhanced the transcription of shelterin components TRF1 and TRF2, as well as DNA repair factor RAD50, conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion. Based on a human single-cell transcriptome atlas, we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation. Using in vitro-cultured human pulmonary endothelial cells, we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans. In addition, the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence. In conclusion, we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging, drawing insights from the longevity of bats.
Hereditary spastic paraplegia (HSP) is a group of genetic motor neuron diseases resulting from length-dependent axonal degeneration of the corticospinal upper motor neurons. Due to the advancement of next-generation sequencing, more than 70 novel HSP disease-causing genes have been identified in the past decade. Despite this, our understanding of HSP physiopathology and the development of efficient management and treatment strategies remain poor. One major challenge in studying HSP pathogenicity is selective neuronal vulnerability, characterized by the manifestation of clinical symptoms that are restricted to specific neuronal populations, despite the presence of germline disease-causing variants in every cell of the patient. Furthermore, disease genes may exhibit ubiquitous expression patterns and involve a myriad of different pathways to cause motor neuron degeneration. In the current review, we explore the correlation between transcriptomic data and clinical manifestations, as well as the importance of interspecies models by comparing tissue-specific transcriptomic profiles of humans and mice, expression patterns of different genes in the brain during development, and single-cell transcriptomic data from related tissues. Furthermore, we discuss the potential of emerging single-cell RNA sequencing technologies to resolve unanswered questions related to HSP pathogenicity.
The biological function of the novel zinc-finger SWIM domain-containing protein family (ZSWIM) during embryonic development remains elusive. Here, we conducted a genome-wide analysis to explore the evolutionary processes of the ZSWIM gene family members in mice, Xenopus tropicalis, zebrafish, and humans. We identified nine putative ZSWIM genes in the human and mouse genome, eight in the Xenopus genome, and five in the zebrafish genome. Based on multiple sequence alignment, three members, ZSWIM5, ZSWIM6, and ZSWIM8, demonstrated the highest homology across all four species. Using available RNA sequencing (RNA-seq) data, ZSWIM genes were found to be widely expressed across different tissues, with distinct tissue-specific properties. To identify the functions of the ZSWIM protein family during embryogenesis, we examined temporal and spatial expression patterns of zswim family genes in Xenopus embryos. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that each member had a distinct expression profile. Whole-mount in situ hybridization showed that both zswim1 and zswim3 were maternally expressed genes; zswim5 and zswim6 were expressed throughout embryogenesis and displayed dynamic expression in the brain, eyes, somite, and bronchial arch at the late tailbud stages; zswim7 was detected in the eye area; zswim8 showed a dynamic expression pattern during the tailbud stages, with expression detected in the brain, eyes, and somite; zswim9 was faintly expressed throughout embryonic development. This study provides a foundation for future research to delineate the functions of ZSWIM gene members.
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