Cross-species Comparisons Research Articles

Cross-species single-cell RNA-seq analysis reveals disparate and conserved cardiac and extracardiac inflammatory responses upon heart injury

The immune system coordinates the response to cardiac injury and controls regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Adult mice and humans lack the ability to fully recover while adult zebrafish spontaneously regenerate after heart injury. Here we profile the inflammatory response to heart cryoinjury in zebrafish and coronary artery ligation in mouse using single cell transcriptomics. We interrogate the extracardiac reaction to cardiomyocyte necrosis to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages play a critical role in determining tissue homeostasis by healing versus scarring. We identify distinct transcriptional clusters of monocytes/macrophages (mono/Mϕ) in each species and find analogous pairs in zebrafish and mice. However, the reaction to myocardial injury is largely disparate between mice and zebrafish. The dichotomous response to heart damage between the murine and zebrafish mono/Mϕ and/or the presence of distinct zebrafish mono/Mϕ subtypes may underlie the impaired regenerative process in adult mammals and humans. Our study furnishes a direct cross-species comparison of immune responses between regenerative and profibrotic myocardial injury models, providing a useful resource to the fields of regenerative biology and cardiovascular research.

MiRNATissueAtlas 2025: an update to the uniformly processed and annotated human and mouse non-coding RNA tissue atlas.

MiRNAs represent a non-coding RNA class that regulate gene expression and pathways. While miRNAs are evolutionary conserved most data stems from Homo sapiens and Mus musculus. As miRNA expression is highly tissue specific, we developed miRNATissueAtlas to comprehensively explore this landscape in H. sapiens. We expanded the H. sapiens tissue repertoire and included M. musculus. In past years, the number of public miRNA expression datasets has grown substantially. Our previous releases of the miRNATissueAtlas represent a great framework for a uniformly pre-processed and label-harmonized resource containing information on these datasets. We incorporate the respective data in the newest release, miRNATissueAtlas 2025, which contains expressions from 9 classes of ncRNA from 799 billion reads across 61 593 samples for H. sapiens and M. musculus. The number of organs and tissues has increased from 28 and 54 to 74 and 373, respectively. This number includes physiological tissues, cell lines and extracellular vesicles. New tissue specificity index calculations build atop the knowledge of previous iterations. Calculations from cell lines enable comparison with physiological tissues, providing a valuable resource for translational research. Finally, between H. sapiens and M. musculus, 35 organs overlap, allowing cross-species comparisons. The updated miRNATissueAtlas 2025 is available at https://www.ccb.uni-saarland.de/tissueatlas2025.

Spatially integrated cortico-subcortical tracing data for analyses of rodent brain topographical organization

The cerebral cortex extends axonal projections to several subcortical brain regions, including the striatum, thalamus, superior colliculus, and pontine nuclei. Experimental tract-tracing studies have shown that these subcortical projections are topographically organized, reflecting the spatial organization of sensory surfaces and body parts. Several public collections of mouse- and rat- brain tract-tracing data are available, with the Allen mouse brain connectivity atlas being most prominent. There, a large body of image data can be inspected, but it is difficult to combine data from different experiments and compare spatial distribution patterns. To enable co-visualization and comparison of topographical organization in mouse brain cortico-subcortical projections across experiments, we represent axonal labelling data as point data in a common 3D brain atlas space. We here present a collection of point-cloud data representing spatial distribution of corticostriatal, corticothalamic, corticotectal, and corticopontine projections in mice and exemplify how these spatially integrated point data can be used as references for experimental investigations of topographic organization in transgenic mice, and for cross-species comparison with corticopontine projections in rats.

Rain-harvesting behavior in free-ranging prairie rattlesnakes (Crotalus viridis)

Abstract Organisms inhabiting arid environments face challenges to obtain dietary water. To prevent desiccation, some organisms possess unique adaptations to harvest water from infrequent and unpredictable rainfall, including several squamates (snakes and lizards). While most squamates consume precipitation as it pools in the environment, a small number engage in behaviors to enhance water collection by capturing precipitation from their own skin, referred to as rain-harvesting behavior (RHB). Details of this behavior remain unclear, particularly the sequence of behaviors associated with RHB. We developed a method to simulate rainfall to observe RHB in prairie rattlesnakes (Crotalus viridis) in situ and recorded 72 events in 94 snakes, the most robust sampling of RHB to date. Using video analysis, we describe the postures and kinematics of RHB and develop the first illustrated ethogram of this behavior for any vertebrate. Our results demonstrate that RHB contains fixed and variable patterns useful in cross-species comparisons and in exploring proximate causes of the behavior. Additionally, we describe novel features of RHB including suspended head drinking, body levering, and drinking from neighboring snakes. Our results reveal RHB to be an intricate suite of movements and actions, some of which allude to acute sensory abilities of these animals that warrant further study. Furthermore, observations of RHB at dens and rookeries suggest a potential, novel benefit of snake aggregation is the formation of large, communal surfaces for rain harvesting. We suggest that the extremely elongated body plan of snakes may be well-suited for a rapidly deployed, modular rain-harvesting system effective at capitalizing on fleeting rainstorms characteristic of arid ecosystems of the world.

IMMU-66. PHYSIOLOGICALLY RELEVANT MURINE GLIOMA MODELS THAT REPRESENT GBM HETEROGENEITY AND ENABLE THE NEXT GENERATION OF DISCOVERY AND THERAPY EFFICACY STUDIES

Abstract To transform cancer drug development pipelines and gain a deeper mechanistic understanding of cancer: immune interactions, we desperately need better experimental models that faithfully recapitulate the cellular heterogeneity, complex and dynamic cell:cell interactions, and therapy responses in humans. To address this need, we developed and characterized a cohort of five new mouse glioma models in the C57BL6/j background. These transplantable tumorsphere models were derived from spontaneous gliomas that formed in genetically engineered mouse models with different oncogenic drivers, including EGFRviii, PDGFA, PTEN, p53, and NF1 mutations. They show a range of responses to standard of care (TMZ+IR) similar to GBM patients and all are resistant, to immune checkpoint inhibitors, anti-PD1+CTLA4, treatment, unlike the GL261 model and similar to GBM patients. We deeply characterized each model, including immune phenotyping and single-cell RNA-sequencing analyses. We also performed a cross-species comparison of human and mouse GBM and stromal cell phenotypes at the single-cell level. Our results show that new mouse models recapitulate the heterogeneity of glioma cell states observed in individual patients and, importantly, also the complex and immune suppressive TME. We demonstrate at the single-cell level that glioma-associated T and myeloid cell types are molecularly similar and conserved across species. Leveraging these data sets and experimental models, we are testing now novel immunotherapy targets for GBM.

Single-cell transcriptomic and cross-species comparison analyses reveal distinct molecular changes of porcine testes during puberty

The pig is an important model for studying human diseases and is also a significant livestock species, yet its testicular development remains underexplored. Here, we employ single-cell RNA sequencing to characterize the transcriptomic landscapes across multiple developmental stages of Bama pig testes from fetal stage through infancy, puberty to adulthood, and made comparisons with those of humans and mice. We reveal an exceptionally early onset of porcine meiosis shortly after birth, and identify a distinct subtype of porcine spermatogonia resembling transcriptome state 0 spermatogonial stem cells identified in humans, which were previously thought to be primate specific. We also discover the persistent presence of proliferating progenitors for myoid cells in postnatal testes. The regulatory roles of Leydig cell steroidogenesis and estrogen synthesis in supporting cell lineages are also explored, including the potential impact of estrogen on Sertoli cell maturation and spermatogenesis. Overall, this study offers valuable insights into porcine testicular development, paving the way for future research in reproductive biology, advancements in agricultural breeding, and potential applications in translational medicine.

High-throughput identification of calcium-regulated proteins across diverse proteomes

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

Adeno-associated viral tools to trace neural development and connectivity across amphibians.

Amphibians, by virtue of their phylogenetic position, provide invaluable insights on nervous system evolution, development, and remodeling. The genetic toolkit for amphibians, however, remains limited. Recombinant adeno-associated viral vectors (AAVs) are a powerful alternative to transgenesis for labeling and manipulating neurons. Although successful in mammals, AAVs have never been shown to transduce amphibian cells efficiently. We screened AAVs in three amphibian species-the frogs Xenopus laevis and Pelophylax bedriagae and the salamander Pleurodeles waltl-and identified at least two AAV serotypes per species that transduce neurons. In developing amphibians, AAVs labeled groups of neurons generated at the same time during development. In the mature brain, AAVrg retrogradely traced long-range projections. Our study introduces AAVs as a tool for amphibian research, establishes a generalizable workflow for AAV screening in new species, and expands opportunities for cross-species comparisons of nervous system development, function, and evolution.

Peripheral blood mononuclear cells isolation from mouse and rabbit blood to quantify active nucleotide and define brincidofovir dose for smallpox.

Aim: Brincidofovir has been approved by the US FDA for the treatment of smallpox via the "Animal Rule". The active moiety, cidofovir diphosphate (CDV-PP), was measurable in human peripheral blood mononuclear cells (PBMCs), but quantitation in animals was challenging given their limited blood volume. The aim of this study was to optimize PBMC isolation from rabbit and mouse blood to allow quantitation of CDV-PP.Materials & methods: PBMC isolation methods using various separation media were evaluated using blood from naive and brincidofovir-dosed animals. CDV-PP was quantified using liquid chromatography tandem mass spectrometry.Results & conclusion: PBMC isolation yields were increased by >fourfold compared with yields obtained using human methods, allowing cross-species exposure comparisons.

Asp/ASPM phospho-regulation throughout the cell cycle.

In mammals and Drosophila melanogaster, Asp/ASPM proteins contribute to cell proliferation and spindle formation. Recent evidence also suggests interphase roles for Asp/ASPM proteins, but little is known about the regulation allowing distinct roles in different cell cycle phases. In this review, we consider a cross-species comparison of Asp/ASPM protein sequences in light of cyclin-cyclin-dependent kinase (CDK) literature, and suggest Asp/ASPM proteins to be prime candidates for cyclin-CDK regulation. Conserved regulatory features include an N-terminal proline directed serine/threonine (S/T-P) "supershift" phosphorylation domain common to proteins with bistable interphase and mitotic roles, as well as putative cyclin-binding sites positioned to allow multisite phosphorylation by cyclin-CDK complexes. Human, mouse, and Drosophila Asp/ASPM protein structural predictions show that multisite phosphorylation of the N-term supershift domain could alter the availability of CH-domains and HEAT-motifs, which can contribute to microtubule binding and protein aggregation likely required for spindle formation. Structural predictions of the smallest reported microcephaly patient truncation also emphasize the importance of the arrangement of these motifs. We position this in silico analysis within recent literature to build new hypotheses for Asp/ASPM regulation in interphase and mitosis, as well as de-regulation in microcephaly and cancer. We also highlight the utility of comparing structural/functional differences between human ASPM and Drosophila Asp to gain further insight.

Comparative transcriptomic rhythms in the mouse and human prefrontal cortex.

Alterations in multiple subregions of the human prefrontal cortex (PFC) have been heavily implicated in psychiatric diseases. Moreover, emerging evidence suggests that circadian rhythms in gene expression are present across the brain, including in the PFC, and that these rhythms are altered in disease. However, investigation into the potential circadian mechanisms underlying these diseases in animal models must contend with the fact that the human PFC is highly evolved and specialized relative to that of rodents. Here, we use RNA sequencing to lay the groundwork for translational studies of molecular rhythms through a sex-specific, cross species comparison of transcriptomic rhythms between the mouse medial PFC (mPFC) and two subregions of the human PFC, the anterior cingulate cortex (ACC) and the dorsolateral PFC (DLPFC). We find that while circadian rhythm signaling is conserved across species and subregions, there is a phase shift in the expression of core clock genes between the mouse mPFC and human PFC subregions that differs by sex. Furthermore, we find that the identity of rhythmic transcripts is largely unique between the mouse mPFC and human PFC subregions, with the most overlap (20%, 236 transcripts) between the mouse mPFC and the human ACC in females. Nevertheless, we find that basic biological processes are enriched for rhythmic transcripts across species, with key differences between regions and sexes. Together, this work highlights both the evolutionary conservation of transcriptomic rhythms and the advancement of the human PFC, underscoring the importance of considering cross-species differences when using animal models.

Cross-species comparison of airway epithelium transcriptomics

Studies of lung transcriptomics across species are essential for understanding the complex biology and disease mechanisms of this vital organ. Single-cell RNA sequencing (scRNA-seq) has emerged as a key tool for understanding cell dynamics across various species. However, comprehensive cross-species comparisons are limited. Therefore, the aims of this study was to investigate the transcriptomic similarities and differences in lung cells across four species—humans, monkeys, mice, and rats—in healthy and asthma conditions using scRNA-seq. The results revealed significant transcriptomic similarities between monkeys and humans and significant cross-species conservation of cell-specific marker genes, transcription factors (TFs), and biological pathways. Additionally, we explored sex differences, identifying distinct sex-specific expression patterns that may influence disease susceptibility. These insights refine our understanding of the mechanism underlying airway cell biology across species and have important implications for studying lung diseases, particularly the mechanisms of mucus clearance in asthma.

Modular, inducible, and titratable expression systems for Escherichia coli and Acinetobacter baumannii.

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii. We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, PabstBR, that induces to a high level but is less leaky than the commonly used trc promoter. We show that PabstBR is titratable at both the population and single-cell levels, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress σ factor, RpoE, and deploy the reporter in E. coli and A. baumannii, finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers who study the fundamental biology of E. coli and A. baumannii.IMPORTANCEAcinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. In this study, we provide expression tools that can be used to study the gene function in A. baumannii, including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli, enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology.

Spatial arrangement of the whiskers of harbor seals ( Phoca vitulina ) compared to whisker arrangements of house mice ( Mus musculus ) and brown rats ( Rattus norvegicus ).

We quantify the three-dimensional positions and orientations of the whiskers across the face of the harbor seal, and compare this geometry with the whisker arrays of rats and mice.

Single-cell RNA sequencing illuminates the ontogeny, conservation and diversification of cartilaginous and bony fish lymphocytes

Elucidating cellular architecture and cell-type evolution across species is central to understanding immune system function and susceptibility to disease. Adaptive immunity is a shared trait of the common ancestor of cartilaginous and bony fishes. However, evolutionary features of lymphocytes in these two jawed vertebrates remain unclear. Here, we present a single-cell RNA sequencing atlas of immune cells from cartilaginous (white-spotted bamboo shark) and bony (zebrafish and Chinese tongue sole) fishes. Cross-species comparisons show that the same cell types across different species exhibit similar transcriptional profiles. In the bamboo shark, we identify a phagocytic B cell population expressing several pattern recognition receptors, as well as a T cell sub-cluster co-expressing both T and B cell markers. In contrast to a division by function in the bony fishes, we show close linkage and poor functional specialization among lymphocytes in the cartilaginous fish. Our cross-species single-cell comparison presents a resource for uncovering the origin and evolution of the gnathostome immune system.

Abstract P367: Cross-Species Epigenomic Maps of Intergenic Loci for Hypertension and Blood Pressure

Many non-coding SNPs identified in Genome-Wide Association Studies (GWAS) likely affect BP-related gene expression through epigenetic mechanisms. This study analyzes the comparative genomic and epigenomic landscapes in human and rat kidney tissues, including kidney proximal tubule (PT) and medullary thick ascending limb (mTAL), focusing on the major challenge of identifying conserved regulatory elements in intergenic regions. The purpose of our study is to identify BP gene regulatory elements in intergenic regions that are conserved from human to rat for in vivo validation and mechanistic studies. Our ultimate goal is to generate high-resolution, genome-wide epigenomic maps of key BP-relevant tissues, using ATAC-seq, Hi-C, CUT&Tag, DNA methylation profiling and RNA-seq. We are creating visualization hubs for human and rat, integrating our data within the UCSC Genome Browser environment. UCSC contains extensive data for humans but very little for rats, particularly for the updated genome assemblies. To address this gap, we integrated our results with data from the Rat Genome Database (RGD). To date, we harmonized ATAC-seq data from human and rat PT and mTAL and displayed these data in RGD’s JBrowse2 genome browser for unique comparative genome views. Based on our initial analysis of data density and intensity, we are developing a novel pattern recognition algorithm to identify epigenomic marks that are conserved across rat and human genomes. Existing algorithms predominantly focus on the conservation of coding genes, gene families, and genomic sequences. Here, average normalized peak intensities for ATAC-seq data across tissues were subject to canonical correlation between human and rat to identify conserved epigenomic patterns within the BP GWAS loci. Here we present an example of a human BP locus in the intergenic region between NPR3 and TARS1 on chromosome 5 which has a region of conserved synteny with rat chromosome 2. Comparative analysis and visualization of this region show significant correlation between ATAC-seq peaks from rat and human PT and mTAL at this locus, with predicted conserved regulatory elements. These visualizations can uncover novel biological insights and identify potential regulatory targets for intervention. This work underscores the importance of advanced visualization techniques in understanding hypertension pathogenesis, facilitating cross-species comparisons, and enhancing the utility of genomic databases.

Brain Charts for the Rhesus Macaque Lifespan.

Recent efforts to chart human brain growth across the lifespan using large-scale MRI data have provided reference standards for human brain development. However, similar models for nonhuman primate (NHP) growth are lacking. The rhesus macaque, a widely used NHP in translational neuroscience due to its similarities in brain anatomy, phylogenetics, cognitive, and social behaviors to humans, serves as an ideal NHP model. This study aimed to create normative growth charts for brain structure across the macaque lifespan, enhancing our understanding of neurodevelopment and aging, and facilitating cross-species translational research. Leveraging data from the PRIMatE Data Exchange (PRIME-DE) and other sources, we aggregated 1,522 MRI scans from 1,024 rhesus macaques. We mapped non-linear developmental trajectories for global and regional brain structural changes in volume, cortical thickness, and surface area over the lifespan. Our findings provided normative charts with centile scores for macaque brain structures and revealed key developmental milestones from prenatal stages to aging, highlighting both species-specific and comparable brain maturation patterns between macaques and humans. The charts offer a valuable resource for future NHP studies, particularly those with small sample sizes. Furthermore, the interactive open resource (https://interspeciesmap.childmind.org) supports cross-species comparisons to advance translational neuroscience research.

MACSFeD-a database of mosquito acoustic communication and swarming features.

Acoustic communication plays an important role during the courtship of many mosquito species. Male mosquitoes show strong attraction to female wing beat frequencies, mediated via spectral matching between female wing beat frequency and male ear mechanical tuning frequency. Such acoustic communication typically occurs within swarms, male-dominated aggregations with species-specific properties. Despite hundreds of relevant publications being available, the lack of a central platform hosting all associated data hinders research efforts and limits cross-species comparisons. Here, we introduce MACSFeD (Mosquito Acoustic Communication and Swarming Features Database), an interactive platform for the exploration of our comprehensive database containing 251 unique reports focusing on different aspects of mosquito acoustic communication, including hearing function, wing beat frequency and phonotaxis, as well as male swarming parameters. MACSFeD serves as an easily accessible, efficient, and robust data visualization tool for mosquito acoustic communication research. We envision that further in-depth studies could arise following the use of this new platform. Database URL: https://minmatt.shinyapps.io/MACSFeD/.

Advancing ecotoxicological studies: Utilizing new approach methodologies to enable cross-species extrapolation and reduce avian testing

Ecological risk assessments of agrochemicals have traditionally depended on in vivo guideline tests using northern bobwhite and mallard to provide relevant endpoints for avian species. However, these studies have limitations, including animal welfare concerns, the time and cost involved, limited potential for extrapolation to more realistic exposure conditions, and the lack of mechanistic understanding. The proof-of-concept work presented a case study for thiamethoxam in three avian species, demonstrating the potential of physiologically based kinetic (PBK) modeling to enable dosimetry extrapolations that inform hazard characterization in risk assessment, and reduce the use of avian testing. The model structure for northern bobwhite and mallard contained ten compartments, while an additional ovulation model was included for chicken in the physiological state of egg-laying. The model was first parameterized and evaluated for chicken and northern bobwhite using in vitro kinetic measurements and in vivo toxicokinetic (TK) data. The chicken model was then extrapolated to mallard based on allometric scaling. The models were then used to map the TK profiles across species by simulating internal dose metrics in different avian toxicology studies. These metrics, including peak blood concentrations (Cmax) and area under the curve (AUC) for blood concentration, were determined for acute, subacute, or chronic toxicity endpoints for mallard and northern bobwhite, enabling a quantitative cross-species and cross-route comparison of dosimetry. The results suggested that the chronic toxicological response of birds exposed to thiamethoxam is highly dependent on internal exposure, while mallard appeared to be more dynamically sensitive to thiamethoxam on an acute oral exposure basis. The case study increases the confidence in using new approach methodologies (NAMs) for interpreting avian toxicity studies and facilitating in vitro-in silico-based ecological risk assessments of agrochemicals.

Cross-species substitution matrix comparison of Tomato leaf curl New Delhi virus (ToLCNDV) with medicinal plant isolates

The main objective of this study was to estimate and compare substitution matrixes of nucleotide frequencies for Tomato leaf curl New Delhi virus (ToLCNDV) with recently identified begomoviral isolates from two medicinal false daisy (Eclipta prostrata) and tomato (Solanum lycopersicum) plants. The ToLCNDV has become a significant limitation to vegetables production in many countries. A polymerase chain reaction was conducted to conserve the existence of begomoviral infection. The acquired amplicon was amplified using primers appropriate to the sequence in order to retrieve the full genome. The sequence analysis has confirmed the presence of ToLCNDV in symptomatic plants. The complete genome sequence having a 2.6–2.7 kb entire genome of ToLCNDV was obtained. An investigation of the phylogenetic and evolutionary history has verified the connection between this virus and other closely related viruses. The available nucleotide frequencies of codon regions (A, T/U, C, G) with newly isolates revealed 20–28% substitution matrixes. There was a minimal difference of nucleotide frequencies’ with already submitted database of this virus. Substitution matrixes, which quantify the probability of nucleotide substitutions evolving over a period of time, offer valuable information about mutation patterns and the forces driving evolution. This comparative analysis enhanced the comprehension of the genetic diversity of ToLCNDV and its possible consequences on medicinal plants. It also assisted in the formulation of efficient control measures and the preservation of begomoviruses in medicinal plant biodiversity. The information presented here is highly valuable for understanding the ToLCNDV biology and epidemiology, and it would also assist in disease management in the future.