Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2.

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.

The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas.

Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 µm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 µm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

TCR Transgenic Mice Reveal Stepwise, Multi-site Acquisition of the Distinctive Fat-Treg Phenotype.

Visceral adipose tissue (VAT) hosts a population of regulatory T (Treg) cells, with a unique phenotype, that controls local and systemic inflammation and metabolism. Generation of a T cell receptor transgenic mouse line, wherein VAT Tregs are highly enriched, facilitated study of their provenance, dependencies, and activities. We definitively established a role for T cell receptor specificity, uncovered an unexpected function for the primordial Treg transcription-factor, Foxp3, evidenced a cell-intrinsic role for interleukin-33 receptor, and ordered these dependencies within a coherent scenario. Genesis of the VAT-Treg phenotype entailed a priming step in the spleen, permitting them to exit the lymphoid organs and surveil nonlymphoid tissues, and a final diversification process within VAT, in response to microenvironmental cues. Understanding the principles of tissue-Treg biology is a prerequisite for precision-targeting strategies.

A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Haploid mouse embryonic stem cells: rapid genetic screening and germline transmission.

Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.

Insights into digit evolution from a fate map study of the forearm using Chameleon, a new transgenic chicken line.

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice.

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

A mammalian tripartite enhancer cluster controls hypothalamic Pomc expression, food intake, and body weight.

Food intake and energy balance are tightly regulated by a group of hypothalamic arcuate neurons expressing the proopiomelanocortin (POMC) gene. In mammals, arcuate-specific POMC expression is driven by two cis-acting transcriptional enhancers known as nPE1 and nPE2. Because mutant mice lacking these two enhancers still showed hypothalamic Pomc mRNA, we searched for additional elements contributing to arcuate Pomc expression. By combining molecular evolution with reporter gene expression in transgenic zebrafish and mice, here, we identified a mammalian arcuate-specific Pomc enhancer that we named nPE3, carrying several binding sites also present in nPE1 and nPE2 for transcription factors known to activate neuronal Pomc expression, such as ISL1, NKX2.1, and ERα. We found that nPE3 originated in the lineage leading to placental mammals and remained under purifying selection in all mammalian orders, although it was lost in Simiiformes (monkeys, apes, and humans) following a unique segmental deletion event. Interestingly, ablation of nPE3 from the mouse genome led to a drastic reduction (>70%) in hypothalamic Pomc mRNA during development and only moderate (<33%) in adult mice. Comparison between double (nPE1 and nPE2) and triple (nPE1, nPE2, and nPE3) enhancer mutants revealed the relative contribution of nPE3 to hypothalamic Pomc expression and its importance in the control of food intake and adiposity in male and female mice. Altogether, these results demonstrate that nPE3 integrates a tripartite cluster of partially redundant enhancers that originated upon a triple convergent evolutionary process in mammals and that is critical for hypothalamic Pomc expression and body weight homeostasis.

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle.

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Contribution of animal models to the mechanistic understanding of Alternative Pathway and Amplification Loop (AP/AL)-driven Complement-mediated Diseases.

This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti-complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease.

Mouse Pramel1 regulates spermatogonial development by inhibiting retinoic acid signaling during spermatogenesis.

Spermatogenesis begins when cell fate-committed prospermatogonia migrate to the basement membrane and initiate spermatogenesis in response to retinoic acid (RA) in the neonatal testis. The underlying cellular and molecular mechanisms in this process are not fully understood. Here, we report findings on the involvement of a cancer/testis antigen, PRAMEL1, in the initiation and maintenance of spermatogenesis. By analyzing mouse models with either global or conditional Pramel1 inactivation, we found that PRAMEL1 regulates the RA responsiveness of the subtypes of prospermatogonia in the neonatal testis, and affects their homing process during the initiation of spermatogenesis. Pramel1 deficiency led to increased fecundity in juvenile males and decreased fecundity in mature males. In addition, Pramel1 deficiency resulted in a regional Sertoli cell-only phenotype during the first round of spermatogenesis, which was rescued by administration of the RA inhibitor WIN18,446, suggesting that PRAMEL1 functions as an inhibitor of RA signaling in germ cells. Overall, our findings suggest that PRAMEL1 fine-tunes RA signaling, playing a crucial role in the proper establishment of the first and subsequent rounds of spermatogenesis.

Glutamate-GABA imbalance mediated by miR-8-5p and its STTM regulates phase-related behavior of locusts.

The aggregation of locusts from solitary to gregarious phases is crucial for the formation of devastating locust plagues. Locust management requires research on the prevention of aggregation or alternative and greener solutions to replace insecticide use, and insect-derived microRNAs (miRNAs) show the potential for application in pest control. Here, we performed a genome-wide screen of the differential expression of miRNAs between solitary and gregarious locusts and showed that miR-8-5p controls the γ-aminobutyric acid (GABA)/glutamate functional balance by directly targeting glutamate decarboxylase (Gad). Blocking glutamate-GABA neurotransmission by miR-8-5p overexpression or Gad RNAi in solitary locusts decreased GABA production, resulting in locust aggregation behavior. Conversely, activating this pathway by miR-8-5p knockdown in gregarious locusts induced GABA production to eliminate aggregation behavior. Further results demonstrated that ionotropic glutamate/GABA receptors tuned glutamate/GABA to trigger/hamper the aggregation behavior of locusts. Finally, we successfully established a transgenic rice line expressing the miR-8-5p inhibitor by short tandem target mimic (STTM). When locusts fed on transgenic rice plants, Gad transcript levels in the brain increased greatly, and aggregation behavior was lost. This study provided insights into different regulatory pathways in the phase change of locusts and a potential control approach through behavioral regulation in insect pests.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

An inducible hACE2 transgenic mouse model recapitulates SARS-CoV-2 infection and pathogenesis in vivo.

The classical manifestation of COVID-19 is pulmonary infection. After host cell entry via human angiotensin-converting enzyme II (hACE2), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus can infect pulmonary epithelial cells, especially the AT2 (alveolar type II) cells that are crucial for maintaining normal lung function. However, previous hACE2 transgenic models have failed to specifically and efficiently target the cell types that express hACE2 in humans, especially AT2 cells. In this study, we report an inducible, transgenic hACE2 mouse line and showcase three examples for specifically expressing hACE2 in three different lung epithelial cells, including AT2 cells, club cells, and ciliated cells. Moreover, all these mice models develop severe pneumonia after SARS-CoV-2 infection. This study demonstrates that the hACE2 model can be used to precisely study any cell type of interest with regard to COVID-19-related pathologies.

Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm (Spodoptera frugiperda), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda. Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb, the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

Visualization and functional characterization of lymphoid organ fibroblasts.

Fibroblastic reticular cells (FRCs) are specialized stromal cells of lymphoid organs that generate the structural foundation of the tissue and actively interact with immune cells. Distinct FRC subsets position lymphocytes and myeloid cells in specialized niches where they present processed or native antigen and provide essential growth factors and cytokines for immune cell activation and differentiation. Niche-specific functions of FRC subpopulations have been defined using genetic targeting, high-dimensional transcriptomic analyses, and advanced imaging methods. Here, we review recent findings on FRC-immune cell interaction and the elaboration of FRC development and differentiation. We discuss how imaging approaches have not only shaped our understanding of FRC biology, but have critically advanced the niche concept of immune cell maintenance and control of immune reactivity.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.