Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

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.

Dynamics of Endothelial Cell Generation and Turnover in Arteries During Homeostasis and Diseases.

BACKGROUND: Endothelial cell (EC) generation and turnover by self-proliferation contributes to vascular repair and regeneration. The ability to accurately measure the dynamics of EC generation would advance our understanding of cellular mechanisms of vascular homeostasis and diseases. However, it is currently challenging to evaluate the dynamics of EC generation in large vessels such as arteries because of their infrequent proliferation. METHODS: By using dual recombination systems based on Cre-loxP and Dre-rox, we developed a genetic system for temporally seamless recording of EC proliferation in vivo. We combined genetic recording of EC proliferation with single-cell RNA sequencing and gene knockout to uncover cellular and molecular mechanisms underlying EC generation in arteries during homeostasis and disease. RESULTS: Genetic proliferation tracing reveals that ≈3% of aortic ECs undergo proliferation per month in adult mice during homeostasis. The orientation of aortic EC division is generally parallel to blood flow in the aorta, which is regulated by the mechanosensing protein Piezo1. Single-cell RNA sequencing analysis reveals 4 heterogeneous aortic EC subpopulations with distinct proliferative activity. EC cluster 1 exhibits transit-amplifying cell features with preferential proliferative capacity and enriched expression of stem cell markers such as Sca1 and Sox18. EC proliferation increases in hypertension but decreases in type 2 diabetes, coinciding with changes in the extent of EC cluster 1 proliferation. Combined gene knockout and proliferation tracing reveals that Hippo/vascular endothelial growth factor receptor 2 signaling pathways regulate EC proliferation in large vessels. CONCLUSIONS: Genetic proliferation tracing quantitatively delineates the dynamics of EC generation and turnover, as well as EC division orientation, in large vessels during homeostasis and disease. An EC subpopulation in the aorta exhibits more robust cell proliferation during homeostasis and type 2 diabetes, identifying it as a potential therapeutic target for vascular repair and regeneration.

Generation of Piezo1-CreER transgenic mice for visualization and lineage tracing of mechanical force responsive cells in vivo.

Cells and tissues are exposed to a wide range of mechanical stimuli during development, tissue homeostasis, repair, and regeneration. Over the past few decades, mechanosensitive ion channels (MSCs), as force-sensing integral membrane proteins, have attracted great attention with regard to their structural dynamics and mechanics at the molecular level and functions in various cells. Piezo-type MSC component 1 (Piezo1) is a newly discovered MSC; it is inherently mechanosensitive. However, which type of cells express Piezo1 in vivo remains unclear. To detect and trace Piezo1-expressing cells, we generated and characterized a novel tamoxifen-inducible Cre knock-in mouse line, Piezo1-CreER, which expresses CreER recombinase under the control of the endogenous Piezo1 promoter. Using this genetic tool, we detected the expression of Piezo1 in various cell types at the embryonic, neonatal, and adult stages. Our data showed that Piezo1 was highly expressed in endothelial cells in all the three stages, while the Piezo1 expression in epithelial cells was dynamic during development and growth. In summary, we established a new genetic tool, Piezo1-CreER, to study Piezo1-expressing cells in vivo during development, injury response, and tissue repair and regeneration.

Role of Cardiac Fibroblasts in Cardiac Injury and Repair.

PURPOSE OF REVIEW: The pathological remodeling of cardiac tissue after injury or disease leads to scar formation. Our knowledge of the role of nonmyocytes, especially fibroblasts, in cardiac injury and repair continues to increase with technological advances in both experimental and clinical studies. Here, we aim to elaborate on cardiac fibroblasts by describing their origins, dynamic cellular states after injury, and heterogeneity in order to understand their role in cardiac injury and repair. RECENT FINDINGS: With the improvement in genetic lineage tracing technologies and the capability to profile gene expression at the single-cell level, we are beginning to learn that manipulating a specific population of fibroblasts could mitigate severe cardiac fibrosis and promote cardiac repair after injury. Cardiac fibroblasts play an indispensable role in tissue homeostasis and in repair after injury. Activated fibroblasts or myofibroblasts have time-dependent impacts on cardiac fibrosis. Multiple signaling pathways are involved in modulating fibroblast states, resulting in the alteration of fibrosis. Modulating a specific population of cardiac fibroblasts may provide new opportunities for identifying novel treatment options for cardiac fibrosis.

Simultaneous quantitative assessment of two distinct cell lineages with a nuclear-localized dual genetic reporter.

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.

Tenascin C+ papillary fibroblasts facilitate neuro-immune interaction in a mouse model of psoriasis.

Dermal fibroblasts and cutaneous nerves are important players in skin diseases, while their reciprocal roles during skin inflammation have not been characterized. Here we identify an inflammation-induced subset of papillary fibroblasts that promotes aberrant neurite outgrowth and psoriasiform skin inflammation by secreting the extracellular matrix protein tenascin-C (TNC). Single-cell analysis of fibroblast lineages reveals a Tnc+ papillary fibroblast subset with pro-axonogenesis and neuro-regulation transcriptomic hallmarks. TNC overexpression in fibroblasts boosts neurite outgrowth in co-cultured neurons, while fibroblast-specific TNC ablation suppresses hyperinnervation and alleviates skin inflammation in male mice modeling psoriasis. Dermal γδT cells, the main producers of type 17 pathogenic cytokines, frequently contact nerve fibers in mouse psoriasiform lesions and are likely modulated by postsynaptic signals. Overall, our results highlight the role of an inflammation-responsive fibroblast subset in facilitating neuro-immune synapse formation and suggest potential avenues for future therapeutic research.

Functional ProTracer identifies patterns of cell proliferation in tissues and underlying regulatory mechanisms.

A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.

Dual genetic tracing reveals a unique fibroblast subpopulation modulating cardiac fibrosis.

After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.

Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.

Organ homeostasis is orchestrated by time- and spatially restricted cell proliferation. Studies identifying cells with superior proliferative capacities often rely on the lineage tracing of a subset of cell populations, which introduces a potential selective bias. In this work, we developed a genetic system [proliferation tracer (ProTracer)] by incorporating dual recombinases to seamlessly record the proliferation events of entire cell populations over time in multiple organs. In the mouse liver, ProTracer revealed more hepatocyte proliferation in distinct zones during liver homeostasis, injury repair, and regrowth. Clonal analysis showed that most of the hepatocytes labeled by ProTracer had undergone cell division. By genetically recording proliferation events of entire cell populations, ProTracer enables the unbiased detection of specific cellular compartments with enhanced regenerative capacities.

A suite of new Dre recombinase drivers markedly expands the ability to perform intersectional genetic targeting.

The use of the dual recombinase-mediated intersectional genetic approach involving Cre-loxP and Dre-rox has significantly enhanced the precision of in vivo lineage tracing, as well as gene manipulation. However, this approach is limited by the small number of Dre recombinase driver constructs available. Here, we developed more than 70 new intersectional drivers to better target diverse cell lineages. To highlight their applicability, we used these new tools to study the in vivo adipogenic fate of perivascular progenitors, which revealed that PDGFRa+ but not PDGFRa-PDGFRb+ perivascular cells are the endogenous progenitors of adult adipocytes. In addition to lineage tracing, we used members of this new suite of drivers to more specifically knock out genes in complex tissues, such as white adipocytes and lymphatic vessels, that heretofore cannot be selectively targeted by conventional Cre drivers alone. In summary, these new transgenic tools expand the intersectional genetic approach while enhancing its precision.