Reporter Mouse Strain Research Articles

IL-12 regulates type 3 immunity through interfollicular keratinocytes in psoriasiform inflammation.

Psoriasis is a chronic inflammatory skin disorder underpinned by dysregulated cytokine signaling. Drugs neutralizing the common p40 subunit of interleukin-12 (IL-12) and IL-23 represented a therapeutic breakthrough; however, new drugs that block the IL-23p19 subunit and spare IL-12 are more effective, suggesting a regulatory function of IL-12. To pinpoint the cell type and underlying mechanism of IL-12–mediated immune regulation in psoriasis, we generated a conditional Il12rb2-knockout (KO)/reporter mouse strain. We detected Il12rb2 expression in T cells and a specific subset of interfollicular (IF) keratinocytes. Analysis of single-cell RNA-sequencing (scRNAseq) data from patients with psoriasis confirmed a similar expression pattern in the human skin. Deletion of Il12rb2 across the hematopoietic compartment did not alter the development of Aldara-induced psoriasiform inflammation. However, depletion of Il12rb2 in keratinocytes exacerbated disease development, phenocopying the Il12rb2 germline knockout. Protective IL-12 signaling blocked the hyperproliferation of keratinocytes, maintained skin barrier integrity, and diminished disease-driving IL-23/type 3 immune circuits. In line, specific IL-23p19 blockade led to a more profound reduction of psoriatic keratinocyte expression signatures in the skin of patients with psoriasis than combined IL-12/IL-23 inhibition. Collectively, we provide a potential explanation for the superior efficacy of IL-23p19 inhibitors in psoriasis and describe an unperceived role of IL-12 in maintaining skin epithelial cell homeostasis.

Generation of a Spiral Ganglion Neuron Degeneration Mouse Model.

Spiral ganglion neurons (SGNs) can be injured by a wide variety of insults. However, there still is a lack of degeneration models to specifically damage the SGNs without disturbing other types of cells in the inner ear. This study aims to generate an SGN-specific damage model using the Cre-LoxP transgenic mouse strains. The Cre-inducible diphtheria toxin receptor (iDTR+/+) knock-in mouse strain was crossed with a mouse strain with Cre activity specific to neurons (NeflCreER/CreER). Expression of the Cre-recombinase activity was evaluated using the reporter mouse strain Ai9 at pre-hearing, hearing onset, and post-hearing stages. Accordingly, heterozygous NeflCreER/+;iDTR+/– mice were treated with tamoxifen on postnatal days 1–5 (P1–5), followed by diphtheria toxin (DT) or vehicle injection on P7, P14, and P21 to evaluate the SGN loss. Robust tamoxifen-induced Cre-mediated Ai9 tdTomato fluorescence was observed in the SGN area of heterozygous NeflCreER/+;Ai9+/– mice treated with tamoxifen, whereas vehicle-treated heterozygote mice did not show tdTomato fluorescence. Compared to vehicle-treated NeflCreER/+;iDTR+/– mice, DT-treated NeflCreER/+;iDTR+/– mice showed significant auditory brainstem response (ABR) threshold shifts and SGN cell loss. Hair cell count and functional study did not show significant changes. These results demonstrate that the NeflCreER/CreER mouse strain exhibits inducible SGN-specific Cre activity in the inner ear, which may serve as a valuable SGN damage model for regeneration research of the inner ear.

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

HLF expression defines the human hematopoietic stem cell state

AbstractHematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages after transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. Although several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population and single-cell transcriptomes of unexpanded and ex vivo cultured cord blood–derived hematopoietic stem and progenitor cells as well as peripheral blood, adult bone marrow, and fetal liver. On the basis of these analyses, we propose the master transcription factor HLF (hepatic leukemia factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter. Most importantly, HLF-expressing cells comprise all stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human HSC state and outline a new approach to continuously mark these cells with high fidelity.

Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line

ObjectivesCone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. MethodsWe generated OPN1LW-EGFP/NRL−/− mice by crossing NRL−/− and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL−/− mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL−/− mice were transplanted subretinally into immunodeficient Rd1 mice. ResultsOPN1LW-EGFP/NRL−/− retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL−/− compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL−/− were similar as the NRL−/− with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL−/− was thinner than NRL−/− controls. The OPN1LW-EGFP/NRL−/− ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL−/− controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL−/− mice showed greater S-opsin + cone survival than suspension grafts from the same strain. ConclusionsOPN1LW-EGFP/NRL−/− retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Imaging of bone and joints in vivo: pathological osteoclastogenesis in arthritis.

Osteoimmunology highlights the reciprocal interactions between the skeletal and immune systems. Over the past two decades, many molecules that link the two have been identified, including cytokines, receptors and transcription factors, leading to successful translation of research into therapeutic approaches to autoimmune diseases such as rheumatoid arthritis. The development of an intravital imaging system using two-photon microscopy, combined with a variety of fluorescent probes and reporter mouse strains, has provided valuable insights into the real-time dynamics of osteoclasts and immune cells in the bone marrow. This technique is now applied to the synovial tissue of arthritic mice to investigate the pathogenesis of osteoimmune diseases and enables direct observation of complex biological phenomena in vivo. In addition, rapid progress in the next-generation sequencing technologies has provided important insights into the field of osteoimmunology through characterizing individual cells in the synovial microenvironment. Single-cell RNA sequencing (scRNA-seq) dissects cellular heterogeneity within a biological system and enables the identification of specific cells differentiating into mature osteoclasts within the previously defined 'osteoclast precursor-containing population'. In this review, we will explain the cellular interactions and cytokine milieu involved in inflammatory bone destruction and update how the novel technologies, such as scRNA-seq and intravital imaging, have contributed to better understand the pathogenesis of bone destruction in arthritis.

Sex dictates organ-specific differences in the production and activation of plasmablasts and plasma cells

Abstract In humans and mice, sex dichotomies exist in regards to physiological immune responses as well as pathological autoimmune responses. However, very little information exists in regards to how sex patterns the production and function of antibody-secreting plasmablast (PB) and plasma cell (PC) populations. Using the Prdm1-enhanced yellow fluorescent (eYFP) reporter mouse strain, we compared the percentages and numbers of PBs and PCs in the bone marrow (BM), spleen (SPL) and thymus (THY) of young (3 months old) female and male mice. While PB/PC generation was equivalent in the BM and SPL of both sexes, the female THY had significantly increased percentages and numbers of both PBs and PCs when compared to males. This correlated with the overall increase in thymopoiesis present in females. Characterization of THY PBs/PCs demonstrated increased expression of canonical B cell activation markers such as CD69 and MHC II when compared to their BM and SPL counterparts. In some aspects, these differences were sex-based in origin. αCD45 intravenous antibody labeling suggested that THY PBs/PCs were locally generated and not a consequence of immigration from the periphery. As such, female THY demonstrated increased numbers of a germinal center B (GCB) cell-like population expressing both GL7 and CD95(Fas). THY B cells have been previously shown to participate in the regulation of T cell selection and we hypothesized that THY PBs/PCs would be generated in a T cell dependent manner. Indeed, administration of αCD40L blocking antibodies ablated THY GCB, PB and to some extent PC production. In summary, female mice are skewed towards increased THY PB/PC generation. The production of these cells happens locally and is dependent on CD40L-based T cell interactions.

The Genetic Basis of Reporter Mouse Strains.

Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

IntroductionFibrocytes are emerging myeloid‐derived circulating cells that can migrate into damaged tissues and usually contribute to their repair. Key features of fibrocytes include the expression myeloid markers, production of extracellular matrix proteins, and secretion of various humoral factors that activate resident fibroblasts; they also have the potential to differentiate into fibroblasts. However, no specific surface markers have been identified to identify fibrocytes in vivo. One reason could be that the method used to detect fibrocytes requires intracellular collagen staining.MethodsIn the present study, to establish an improved method for the detection of lung fibrocytes and to analyze viable fibrocytes, we used collagen I(α)2‐green fluorescent protein (Col‐GFP) reporter mice, which had undergone the intratracheal instillation of bleomycin (BLM).ResultsUsing flow cytometry to gate out cells with autofluorescence, we clearly found that CD45+ GFP+ cells resided in the lungs of Col‐GFP mice at a steady state and these cells increased after BLM injury, peaking at Day 14. These cells expressed not only known cell surface markers of fibrocytes, but also some novel markers, in addition to a low level of collagen I in comparison to CD45− GFP+ cells.ConclusionOur findings suggest that the improved method can be a useful for the detection of pure lung fibrocytes and allows us to further analyze the characteristics of viable fibrocytes.

The Development of Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium.

ABSTRACTConsiderable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP‐cerulean/DMP1‐cherry) mouse strain and mice with ectopic enamel (Spry2+/−;Spry4−/−), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel‐facing dentin. In the odontoblasts, which produce enamel‐facing dentin, we identified uniquely expressed genes (Dkk1, Wisp1, and Sall1) that were either absent or downregulated in odontoblasts, which form cementum‐facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Formation and regeneration of a Wnt-responsive junctional epithelium.

To identify the molecular mechanisms mediating the persistent defensive functions of the self-renewing junctional epithelium (JE). Two strains of Wnt reporter mice, Axin2CreErt2/+ ;R26RmTmG/+ and Axin2LacZ/+ , were employed, along with three clinically relevant experimental scenarios where the function of the JE is disrupted: after tooth extraction, after a partial gingivectomy, and after a complete circumferential gingivectomy. Using transgenic Wnt reporter strains of mice, we established the JE is a Wnt-responsive epithelium beginning at the time of its formation and that it maintains this status into adulthood. After tooth extraction, progeny of the initial Wnt-responsive JE population directly contributed to healing and ultimately adopted an oral epithelium (OE) phenotype. In the traditional partial gingivectomy model, the JE completely regenerated and did so via progeny of the original Wnt-responsive population. However, following circumferential gingivectomy, the OE was incapable of re-establishing a functional JE. A Wnt-responsive niche at the interface between tooth and oral epithelia is required for a functional JE.

Adipose Stromal Cells Enhance Decellularized Adipose Tissue Remodeling Through Multimodal Mechanisms.

Decellularized adipose tissue (DAT) scaffolds represent a promising cell-instructive platform for soft tissue engineering. While recent work has highlighted that mesenchymal stromal cells, including adipose-derived stromal cells (ASCs), can be combined with decellularized scaffolds to augment tissue regeneration, the mechanisms involved require further study. The objective of this work was to probe the roles of syngeneic donor ASCs and host-derived macrophages in tissue remodeling of DAT scaffolds within an immunocompetent mouse model. Dual transgenic reporter mouse strains were employed to track and characterize the donor ASCs and host macrophages within the DAT implants. More specifically, ASCs isolated from dsRed mice were seeded on DAT scaffolds, and the seeded and unseeded control scaffolds were implanted subcutaneously into MacGreen transgenic mice for up to 8 weeks. ASC seeding was shown to augment cell infiltration into the DAT implants at 8 weeks, and this was linked to significantly enhanced angiogenesis relative to the unseeded controls. Immunohistochemical staining demonstrated long-term retention of the syngeneic donor ASCs over the duration of the 8-week study, providing evidence that the DAT scaffolds are a cell-supportive delivery platform. Notably, newly formed adipocytes within the DAT implants were not dsRed+, indicating that the donor ASCs supported fat formation through indirect mechanisms. Immunohistochemical tracking of host macrophages through costaining for enhanced green fluorescent protein with the macrophage marker Iba1 revealed that ASC seeding significantly increased the number of infiltrating macrophages within the DAT implants at 3 weeks, while the fraction of macrophages relative to the total cellular infiltrate was similar between the groups at 1, 3, and 8 weeks. Consistent with the tissue remodeling response that was observed, western blotting demonstrated that there was significantly augmented expression of CD163 and CD206, markers of constructive M2-like macrophages, within the ASC-seeded DAT implants. Overall, our results demonstrate that exogenous ASCs enhance tissue regeneration within DAT scaffolds indirectly through multimodal mechanisms that include host cell recruitment and immunomodulation. These data provide further evidence to support the use of decellularized scaffolds as a delivery platform for ASCs in tissue engineering.

MHC-Independent Thymic Selection of CD4 and CD8 Coreceptor Negative αβ T Cells.

It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4-CD8-TCRαβ+, double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5high DN thymocytes that preferentially yield TCRαβ+ CD8αα intestinal lymphocytes, we find that mature CD5low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4+CD8+ (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells.

Limitations of the GENSAT Egr1-EGFP transgenic mouse strain for neural circuit activity mapping

Transgenic immediate-early gene reporter mouse strains are valuable tools for studying activity-dependent neural cell populations in vivo. However, routine characterization of the Gene Expression Nervous System Atlas (GENSAT) “Egr1-EGFP” reporter mouse strain produced results that were highly inconsistent with endogenous Egr1 expression. Activity-dependent EGFP expression was not observed, and EGFP protein did not co-localize with native Egr1 protein. This precautionary study outlines the limitations of the Egr1-EGFP transgenic line as a tool to study the activity-dependent expression of Egr1 and emphasizes the necessity of taking into account the potential loss of regulatory elements, stability determinants, or translational modulation in transgenic reporter strains.

A Critical Blimp-1-Dependent IL-10 Regulatory Pathway in T Cells Protects From a Lethal Pro-inflammatory Cytokine Storm During Acute Experimental Trypanosoma brucei Infection.

In many infectious diseases, the immune response operates as a double-edged sword. While required for protective immunity, infection-induced inflammation can be detrimental if it is not properly controlled, causing collateral body damage and potentially leading to death. It is in this context that the potent anti-inflammatory cytokine interleukin-10 (IL-10) is required to dampen the pro-inflammatory immune response that hallmarks trypanosomosis. Effective control of this infection requires not just the action of antibodies specific for the parasite's variable surface glycoprotein (VSG) coat antigens, but also a pro-inflammatory immune response mediated mainly by IFNγ, TNF, and NO. However, strict control of inflammation is mandatory, as IL-10-deficient mice succumb from an unrestrained cytokine storm within 10 days of a Trypanosome brucei infection. The relevant cellular source of IL-10 and the associated molecular mechanisms implicated in its trypanosomosis associated production are poorly understood. Using an IL-10 reporter mouse strain (Vert-X), we demonstrate here that NK cells, CD8+ T cells and CD4+ T cells as well as B cells and plasma cells constitute potential cellular sources of IL-10 within the spleen and liver during acute infection. The IL-10 wave follows peak pro-inflammatory cytokine production, which accompanied the control of peak parasitemia. Similar results were observed following conventional experimental needle infection and physiological infections via T. brucei-infected tsetse flies. Our results show that conditional T cell-specific ablation of the IL-10 regulating Prdm1 gene (encoding for the Blimp-1 transcription factor), leads to an uncontrolled trypanosome-induced pro-inflammatory syndrome like the one observed in infected IL-10-deficient mice. This result indicates that the biological role of IL-10-derived from non-T cells, including NK cells, is of minor importance when considering host survival. The cytokine IL-27 that is also considered to be an IL-10 regulator, did not affect IL-10 production during infection. Together, these data suggest that T. brucei activates a Blimp-1-dependent IL-10 regulatory pathway in T cells that acts as a critical anti-inflammatory rheostat, mandatory for host survival during the acute phase of parasitemia.

Slug-expressing mouse prostate epithelial cells have increased stem cell potential.

Deciphering the properties of adult stem cells is crucial for understanding of their role in healthy tissue and in cancer progression as well. Both stem cells and cancer stem cells have shown association with epithelial-to-mesenchymal transition (EMT) in various tissue types. Aiming to investigate the epithelial and mesenchymal phenotypic traits in adult mouse prostate, we sorted subpopulations of basal prostate stem cells (mPSCs) and assessed the expression levels of EMT regulators and markers with custom-designed gene expression array. The population of mPSCs defined by a Lin-/Sca-1+CD49fhi/Trop-2+ (LSC Trop-2+) surface phenotype was enriched in mesenchymal markers, especially EMT master regulator Slug, encoded by the Snai2 gene. To further dissect the role of Slug in mPSCs, we used transgenic Snai2tm1.1Wbg reporter mouse strain. Using this model, we confirmed the presence of mesenchymal traits and increase of organoid forming capacity in Slug+ population of mPSCs. The Slug+-derived organoids comprised all prostate epithelial cell types - basal, luminal, and neuroendocrine. Collectively, these data uncover the important role of Slug expression in the physiology of mouse prostate stem cells.

Novel, Non-Gene-Destructive Knock-In Reporter Mice Refute the Concept of Monoallelic Gata3 Expression.

Accurately tuned expression levels of the transcription factor GATA-3 are crucial at several stages of T cell and innate lymphoid cell development and differentiation. Moreover, several lines of evidence suggest that Gata3 expression might provide a reliable molecular marker for the identification of elusive progenitor cell subsets at the earliest stages of T lineage commitment. To be able to faithfully monitor Gata3 expression noninvasively at the single-cell level, we have generated a novel strain of knock-in reporter mice, termed GATIR, by inserting an expression cassette encoding a bright fluorescent marker into the 3'-untranslated region of the endogenous Gata3 locus. Importantly, in contrast to three previously published strains of Gata3 reporter mice, GATIR mice preserve physiological Gata3 expression on the targeted allele. In this study, we show that GATIR mice faithfully reflect endogenous Gata3 expression without disturbing the development of GATA-3-dependent lymphoid cell populations. We further show that GATIR mice provide an ideal tool for noninvasive monitoring of Th2 polarization and straightforward identification of innate lymphoid cell 2 progenitor populations. Finally, as our reporter is non-gene-destructive, GATIR mice can be bred to homozygosity, not feasible with previously published strains of Gata3 reporter mice harboring disrupted alleles. The availability of hetero- and homozygous Gata3 reporter mice with an exceptionally bright fluorescent marker, allowed us to visualize allelic Gata3 expression in individual cells simply by flow cytometry. The unambiguous results obtained provide compelling evidence against previously postulated monoallelic Gata3 expression in early T lineage and hematopoietic stem cell subsets.

Follicle architects in the colonic mucosa

Macrophages Experimental Salmonella infections in the colons of mice are associated with a heterogeneous infiltrate of inflammatory myeloid cells, including a predominance of cells displaying the chemokine receptor CX3CR1. Koscso et al. used a fluorescent reporter mouse strain to separate the inflammatory myeloid infiltrate into subsets of cells with low, intermediate, and high levels of CX3CR1. RNA-sequencing analysis provided clues to the functional roles of each subset. Macrophages with high CX3CR1 were enriched within tertiary lymphoid structures that developed at mucosal sites of Salmonella invasion and contributed to the induction of localized immunoglobulin A antibody responses to Salmonella . These findings enhance our understanding of the functional diversification of the macrophage subsets associated with colonic inflammation. Sci. Immunol. 5 , eaax0062 (2020).

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Deletion of M-Opsin Prevents M Cone Degeneration in a Mouse Model of Leber Congenital Amaurosis

Mutations in retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal M/long-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat-/- mice exhibit proteasome stress because of the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents M cone degeneration in Lrat-/-Opn1sw-/- mice (a pure M cone LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in M cone degeneration in Lrat-/- model. This finding may represent a general mechanism for M cone degeneration in multiple forms of cone degeneration because of M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slowphotoreceptor degeneration.