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Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

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 p16 Ink4a -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 p16 Ink4a + 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 p16 Ink4a + macrophages significantly mitigates hepatocellular damage, whereas eliminating p16 Ink4a + ECs aggravates liver injury. Additionally, targeted reprogramming of p16 Ink4a + ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16 Ink4a + cells and offers insights for developing cell-type-specific senolytic therapies in the future. [Display omitted] • Intersectional genetics enables tracing, ablation, and manipulation of defined p16 Ink4a + cell types • Ablation of p16 Ink4a + macrophages ameliorates liver fibrosis • p16 Ink4a + endothelial cells play a reparative role in liver injury • Reprogramming of p16 Ink4a + endothelial cells ameliorates liver fibrosis A dual recombinase-mediated genetic system for cell-type-specific lineage tracing, ablation, and gene manipulation of senescent cells reveals distinct roles of senescence across cell types. [ABSTRACT FROM AUTHOR]

Dedifferentiated adipose tissue (DFAT) has been proposed as a promising source of patient-specific multipotent progenitor cells (MPPs). During induced dedifferentiation, adipocytes exhibit profound gene expression and cell morphology changes. However, dedifferentiation of post-mitotic cells is expected to enable proliferation, which is critical if enough MPPs are to be obtained. Here, lineage tracing was employed to quantify cell proliferation in mouse adipocytes subjected to a dedifferentiation-inducing protocol commonly used to obtain DFAT cells. No evidence of cell proliferation in adipocyte-derived cells was observed, in contrast to the robust proliferation of non-adipocyte cells present in adipose tissue. We conclude that proliferative MPPs derived using the ceiling culture method most likely arise from non-adipocyte cells in adipose tissue. [ABSTRACT FROM AUTHOR]

Following previous editions, the fifteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) on Methods in Mammary Gland Biology and Breast Cancer was held from the 2nd to the 4th of May in Weggis, Switzerland. Over the course of this meeting, participants followed and discussed presentations from a roster of internationally renowned invited speakers and selected abstracts, complemented with two poster sessions covering exciting unpublished results. The sessions covered projects on normal mammary gland development, breast cancer evolution and metastasis, as well as epigenetic and metabolic regulation of breast cancer. As last year, early career researchers (ECR) hosted a pre-workshop day, when a stage was provided to allow PhD students and postdocs to showcase their projects and results, and when they had ample time for discussions on experimental settings and career planning, while interacting with several invited guests. [ABSTRACT FROM AUTHOR]

Stroke is a prominent contributor to mortality and impairment on a global scale. Ischemic stroke accounts for approximately 80% of stroke cases and is caused by occlusion of cerebral blood vessels. Enhancing neurogenesis through the modulation of the neural stem cell niche in the adult brain is a promising therapeutic strategy for individuals afflicted with ischemic stroke. Neurogenesis results in the generation of newborn neurons that serve as replacements for deceased neural cells within the ischemic core, thereby playing a significant role in the process of neural restoration subsequent to cerebral ischemia. Research has shown that activation of the Wnt/β-catenin pathway can augment neurogenesis following cerebral ischemia, suggesting that this pathway is a potentially beneficial therapeutic target for managing ischemic stroke. This review provides an extensive analysis of the current knowledge regarding the involvement of the Wnt/β-catenin pathway in promoting neurogenesis, thereby offering a promising avenue for therapeutic intervention in the context of ischemic stroke or other neurological impairments. [ABSTRACT FROM AUTHOR]

Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single‐Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent‐lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation‐bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa‐generated tracking trajectory to improve the accuracy of pseudo‐time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. Key points: SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function.The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness.The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent‐lineage differentiation.The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo.The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset.SCALeBa improves the accuracy of differentiation trajectories in monocle2‐based pseudo‐time analysis. [ABSTRACT FROM AUTHOR]

Establishing a highly efficient photoactivatable Cre recombinase PA‐Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light‐mediated optical regulation of Cre‐loxP recombination using PA‐Cre3.0 transgenic early mouse pre‐implantation embryos. We found that inducing PA‐Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA‐Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA‐Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos. [ABSTRACT FROM AUTHOR]

Cell tracking in microscopy images is fundamental to new biological and medical discoveries today. It facilitates the study of the properties of living cells over time. Due to the temporal nature of the cell tracking task, data association is the most difficult aspect of automated cell tracking. Due to the intricate nature of biological, imaging, and algorithmic factors that influence cell segmentation and tracking results, it is challenging to provide a simple and efficient method to determine the appropriate approach for a specific dataset. For example, mitosis, a crucial biological process, plays a key role in correcting trajectories. This research looked at all the challenges of the cell tracking task and the current solutions that have been proposed so far. In this paper, we carefully identified the sources of the tracking challenges and categorized them in a hierarchical diagram to explain the impact of challenges at different levels on the different cell tracking subtasks. Then, after identifying the solutions provided so far, we classified them into three levels: strategic, tactical, and technical. At the strategy level, tracking before detection and tracking by detection are two main approaches. The tactics can be based on cell distance, similarity, overlap, motions, probability, model evaluation, and deep-learning methods. The techniques identified in our analysis include contour evolution, nearest-neighbor linking, morphological-operator-based tracking, similarity-based label propagation, overlap-based label propagation, motion-prediction-based label propagation, graph-based multiple hypothesis tracking, probability-graph-based global optimization, probability-model-based global optimization, and recently developed deep-learning models. By merging cell tracking methods at different levels in one diagram, this classification will help to understand current solutions and provide new insights for cell tracking algorithms. Overall, in this study, we conducted a comprehensive investigation of the challenges of cell tracking and its corresponding solutions, offering a unique source of information. [ABSTRACT FROM AUTHOR]

Cell plasticity describes the ability of cells to alter their phenotypes in response to external stimuli without changing their genotypes. Cell plasticity is essential for metazoan tissue biology and has significant clinical implications. The study of cell plasticity requires experimental models that are amenable to systematic functional perturbations. Single-cell technologies enable the study of cell plasticity across multiple modalities, allowing the construction of functional plasticity phenoscapes. Metazoan organisms are heterocellular systems composed of hundreds of different cell types, which arise from an isogenic genome through differentiation. Cellular 'plasticity' further enables cells to alter their fate in response to exogenous cues and is involved in a variety of processes, such as wound healing, infection, and cancer. Recent advances in cellular model systems, high-dimensional single-cell technologies, and lineage tracing have sparked a renaissance in plasticity research. Here, we discuss the definition of cell plasticity, evaluate state-of-the-art model systems and techniques to study cell-fate dynamics, and explore the application of single-cell technologies to obtain functional insights into cell plasticity in healthy and diseased tissues. The integration of advanced biomimetic model systems, single-cell technologies, and high-throughput perturbation studies is enabling a new era of research into non-genetic plasticity in metazoan systems. [ABSTRACT FROM AUTHOR]

A recent study published in Cell by Liu et al. introduced a new dual homologous recombinase‐mediated genetic profiling tracer technique to track lung epithelial cells accurately, shedding light on the regenerative origin and regulatory mechanism of adult alveolar epithelial stem cells. The research highlights the importance of alveolar regeneration for maintaining lung homeostasis and facilitating post‐injury healing, emphasizing the role of club cells and bronchioalveolar stem cells in promoting alveolar regeneration after lung injury. By developing a novel dual homologous recombination tool, the authors were able to identify club cells as the primary source of alveolar stem cell regeneration, offering new insights for lung stem cell therapy and potential treatment approaches for lung damage healing. [Extracted from the article]

Taste buds, the neuroepithelial organs responsible for the detection of gustatory stimuli in the oral cavity, arise from stem/progenitor cells among nearby basal keratinocytes. Using genetic lineage tracing, Lgr5 and Lgr6 were suggested as the specific markers for the stem/progenitor cells of taste buds, but recent evidence implied that taste buds may arise even in the absence of these markers. Thus, we wanted to verify the genetic lineage tracing of lingual Lgr5- and Lgr6-expressing cells. Unexpectedly, we found that antibody staining revealed more diverse Lgr5-expressing cells inside and outside the taste buds of circumvallate papillae than was previously suggested. We also found that, while tamoxifen-induced genetic recombination occurred only in cells expressing the Lgr5 reporter GFP, we did not see any increase in the number of recombined daughter cells induced by consecutive injections of tamoxifen. Similarly, we found that cells expressing Lgr6, another stem/progenitor cell marker candidate and an analog of Lgr5, also do not generate recombined clones. In contrast, Lgr5-expressing cells in fungiform papillae can transform into Lgr5-negative progeny. Together, our data indicate that lingual Lgr5- and Lgr6-expressing cells exhibit diversity in their capacity to transform into Lgr5- and Lgr6-negative cells, depending on their location. Our results complement previous findings that did not distinguish this diversity.

Establishing a highly efficient photoactivatable Cre recombinase PA‐Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light‐mediated optical regulation of Cre‐loxP recombination using PA‐Cre3.0 transgenic early mouse pre‐implantation embryos. We found that inducing PA‐Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA‐Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA‐Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Abstract Simultaneous profiling of single-cell gene expression and lineage history holds enormous potential for studying cellular decision-making. Recent computational approaches combine both modalities into cellular trajectories; however, they cannot make use of all available lineage information in destructive time-series experiments. Here, we present moslin, a Gromov-Wasserstein-based model to couple cellular profiles across time points based on lineage and gene expression information. We validate our approach in simulations and demonstrate on Caenorhabditis elegans embryonic development how moslin predicts fate probabilities and putative decision driver genes. Finally, we use moslin to delineate lineage relationships among transiently activated fibroblast states during zebrafish heart regeneration.

Abstract The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRβ-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single‐cell RNA‐sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R‐GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi‐potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56− quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF‐A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes. [ABSTRACT FROM AUTHOR]

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/−;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells. NEW & NOTEWORTHY: Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy. [ABSTRACT FROM AUTHOR]

The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRβ-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization. [ABSTRACT FROM AUTHOR]

Tissue-resident stem cells are essential for development and repair, and in the skeleton, this function is fulfilled by recently identified skeletal stem cells (SSCs). However, recent work has identified that SSCs are not monolithic, with long bones, craniofacial sites, and the spine being formed by distinct stem cells. Recent studies have utilized techniques such as fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing to investigate the involvement of SSCs in bone development, homeostasis, and disease. These investigations have allowed researchers to map the lineage commitment trajectory of SSCs in different parts of the body and at different time points. Furthermore, recent studies have shed light on the characteristics of SSCs in both physiological and pathological conditions. This review focuses on discussing the spatiotemporal distribution of SSCs and enhancing our understanding of the diversity and plasticity of SSCs by summarizing recent discoveries. [ABSTRACT FROM AUTHOR]

Single-cell RNA-seq (scRNA-seq) is a revolutionary technology that allows for the genomic investigation of individual cells in a population, allowing for the discovery of unusual cells associated with cancer and metastasis. ScRNA-seq has been used to discover different types of cancers with poor prognosis and medication resistance such as lung cancer, breast cancer, ovarian cancer, and gastric cancer. Besides, scRNA-seq is a promising method that helps us comprehend the biological features and dynamics of cell development, as well as other disorders. This review gives a concise summary of current scRNA-seq technology. We also explain the main technological steps involved in implementing the technology. We highlight the present applications of scRNA-seq in cancer research, including tumor heterogeneity analysis in lung cancer, breast cancer, and ovarian cancer. In addition, this review elucidates potential applications of scRNA-seq in lineage tracing, personalized medicine, illness prediction, and disease diagnosis, which reveals that scRNA-seq facilitates these events by producing genetic variations on the single-cell level. [ABSTRACT FROM AUTHOR]

The central nervous system (CNS) is a complicated neural network. The origin and evolution of functional neurons and glia cells remain unclear, as do the cellular alterations that occur during the course of cerebral disease rehabilitation. Lineage tracing is a valuable method for tracing specific cells and achieving a better understanding of the CNS. Recently, various technological breakthroughs have been made in lineage tracing, such as the application of various combinations of fluorescent reporters and advances in barcode technology. The development of lineage tracing has given us a deeper understanding of the normal physiology of the CNS, especially the pathological processes. In this review, we summarize these advances of lineage tracing and their applications in CNS. We focus on the use of lineage tracing techniques to elucidate the process CNS development and especially the mechanism of injury repair. Deep understanding of the central nervous system will help us to use existing technologies to diagnose and treat diseases. [ABSTRACT FROM AUTHOR]

Cell replacement therapy represents a promising approach for treating neurodegenerative diseases. Contrary to the common addition strategy to generate new neurons from glia by overexpressing a lineage-specific transcription factor(s), a recent study introduced a subtraction strategy by depleting a single RNA-binding protein, Ptbp1, to convert astroglia to neurons not only in vitro but also in the brain. Given its simplicity, multiple groups have attempted to validate and extend this attractive approach but have met with difficulty in lineage tracing newly induced neurons from mature astrocytes, raising the possibility of neuronal leakage as an alternative explanation for apparent astrocyte-to-neuron conversion. This review focuses on the debate over this critical issue. Importantly, multiple lines of evidence suggest that Ptbp1 depletion can convert a selective subpopulation of glial cells into neurons and, via this and other mechanisms, reverse deficits in a Parkinson's disease model, emphasizing the importance of future efforts in exploring this therapeutic strategy.

Dedifferentiated adipose tissue (DFAT) has been proposed as a promising source of patient-specific multipotent progenitor cells (MPPs). During induced dedifferentiation, adipocytes exhibit profound gene expression and cell morphology changes. However, dedifferentiation of post-mitotic cells is expected to enable proliferation, which is critical if enough MPPs are to be obtained. Here, lineage tracing was employed to quantify cell proliferation in mouse adipocytes subjected to a dedifferentiation-inducing protocol commonly used to obtain DFAT cells. No evidence of cell proliferation in adipocyte-derived cells was observed, in contrast to the robust proliferation of non-adipocyte cells present in adipose tissue. We conclude that proliferative MPPs derived using the ceiling culture method most likely arise from non-adipocyte cells in adipose tissue.

Abstract Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single‐Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent‐lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation‐bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa‐generated tracking trajectory to improve the accuracy of pseudo‐time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. Key points SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function. The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness. The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent‐lineage differentiation. The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo. The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset. SCALeBa improves the accuracy of differentiation trajectories in monocle2‐based pseudo‐time analysis.

Abstract Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (

Transit-amplifying (TA) cells are progenitors that undergo an amplification phase followed by transition into an extinction phase. A long postulated epidermal TA progenitor with biphasic behavior has not yet been experimentally observed in vivo. Here, we identify such a TA population using clonal analysis of Aspm-CreER genetic cell-marking in mice, which uncovers contribution to both homeostasis and injury repair of adult skin. This TA population is more frequently dividing than a Dlx1-CreER-marked long-term self-renewing (e.g. stem cell) population. Newly developed generalized birth-death modeling of long-term lineage tracing data shows that both TA progenitors and stem cells display neutral competition, but only the stem cells display neutral drift. The quantitative evolution of a nascent TA cell and its direct descendants shows that TA progenitors indeed amplify the basal layer before transition and that the homeostatic TA population is mostly in extinction phase. This model will be broadly useful for analyzing progenitors whose behavior changes with their clone age. This work identifies a long-missing class of non-self-renewing biphasic epidermal TA progenitors and has broad implications for understanding tissue renewal mechanisms. [ABSTRACT FROM AUTHOR]

Epicardial cells regulate heart growth by secreting numerous growth factors and undergoing lineage specification into other cardiac lineages. However, the lack of specificmarker genes for epicardial cells has hindered the understanding of this cell type in heart development. Through the analysis of a cardiac single cell mRNA sequencing dataset, we identified a novel epicardial gene named Keratin 19 (Krt19). Further analysis of the expression patterns of Krt19 and Wt1, a well-known epicardial gene, revealed their preferences in major cardiac cell types. Using lineage-tracing analysis, we analyzed Krt19-CreER labeled cells at multiple time windows and found that it labels epicardial cells at both embryonic and neonatal stages. Furthermore, we studied the function of epicardial cells using a diphtheria toxin A chain (DTA)-based cell ablation system. We discovered that Krt19-CreER labeled cells are essential for fetal heart development. Finally, we investigated the function of Krt19-CreER and Wt1-CreER labeled cells in neonatal mouse development. We observed that the Krt19-CreER; Rosa-DTA mice displayed a smaller size after tamoxifen treatment, suggesting the potential importance of Krt19-CreER labeled cells in neonatal mouse development. Additionally, we found that Wt1-CreER; Rosa-DTA mice died at early stages, likely due to defects in the kidney and spleen. In summary, we have identified Krt19 as a new epicardial cell marker gene and further explored the function of epicardial cells using the Krt19-CreER and Wt1-CreER-mediated DTA ablation system. [ABSTRACT FROM AUTHOR]

Jawbones and long bones, despite their shared skeletal lineage, frequently exhibit distinct origins and developmental pathways. Identifying specific progenitor subsets for mandibular osteogenesis remains challenging. Type II collagen is conventionally associated with cartilaginous structures, yet our investigation has identified the presence of type II collagen positive (Col2+) cells within the jawbone development and regeneration. The role of Col2+ cells in jawbone morphogenesis and repair has remained enigmatic. In this study, we analyze single-cell RNA sequencing data from mice jawbone at embryonic day 10.5. Through fate-mapping experiments, we have elucidated that Col2+ cells and their progeny are instrumental in mandibular osteogenesis across both fetal and postnatal stages. Furthermore, lineage tracing with a tamoxifen-inducible CreER system has established the pivotal role of Col2+ cells, marked by Col2-CreER and originating from the primordial Meckel's cartilage, in jawbone formation. Moreover, our research explored models simulating jawbone defects and tooth extraction, which underscored the osteogenic differentiation capabilities of postnatal Col2+ cells during repair. This finding not only highlights the regenerative potential of Col2+ cells but also suggests their versatility in contributing to skeletal healing and regeneration. In conclusion, our findings position Col2+ cells as essential in orchestrating osteogenesis throughout the continuum of mandibular development and repair. [ABSTRACT FROM AUTHOR]

Introduction: Hox genes govern rostro-caudal identity along the developing spinal cord, which has a well-defined division of function between dorsal (sensory) and ventral (motor) halves. Here we exploit developmental Hoxb8 expression, normally restricted to the dorsal cord below the obex, to genetically label spinal cord-to-brain ("spinofugal") axons. Methods: We crossed two targeted (knock-in) and two non-targeted recombinase-expressing lines (Hoxb8-IRES-Cre and Hoxb8-T2AFlpO; Hoxb8- Cre and Hoxb8-FlpO, respectively) with appropriate tdtomato-expressing reporter strains. Serial sectioning, confocal and superresolution microscopy, as well as light-sheet imaging was used to reveal robust labeling of ascending axons and their terminals in expected and unexpected regions. Results: This strategy provides unprecedented anatomical detail of ascending spinal tracts anterior to the brainstem, and reveals a previously undescribed decussating tract in the ventral hypothalamus (the spinofugal hypothalamic decussating tract, or shxt). The absence of Hoxb8-suppressing elements led to multiple instances of ectopic reporter expression in Hoxb8-Cre mice (retinal ganglion and vomeronasal axons, anterior thalamic nuclei and their projections to the anterior cingulate and retrosplenial cortices and subiculum, and a population of astrocytes at the cephalic flexure) and Hoxb8-FlpO mice (Cajal--Retzius cells of the dentate gyrus, and mesenchymal cells of the choroid plexus). While targeted transgenic lines were similar in terms of known spinofugal projections, Hoxb8- IRES-Cre reporters had an additional projection to the core of the facial motor nucleus, and more abundant Hoxb8-lineage microglia scattered throughout the brain than Hoxb8-T2A-FlpO (or any other) mice, suggesting dysregulated Hoxb8- driven reporter expression in one or both lines. Discussion: This work complements structural and connectivity atlases of the mouse central nervous system, and provides a platform upon which their reactions to injury or disease can be studied. Ectopic Hoxb8-driven recombinase expression may also be a useful tool to study structure and function of other cell populations in non-targeted lines. [ABSTRACT FROM AUTHOR]

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood–brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells. [ABSTRACT FROM AUTHOR]

Liver kinase B1 (Lkb1), encoded by serine/threonine kinase (Stk11), is a serine/threonine kinase and tumor suppressor that is strongly implicated in Peutz–Jeghers syndrome (PJS). Numerous studies have shown that mesenchymal‐specific Lkb1 is sufficient for the development of PJS‐like polyps in mice. However, the cellular origin and components of these Lkb1‐associated polyps and underlying mechanisms remain elusive. In this study, we generated tamoxifen‐inducible Lkb1flox/flox;Myh11‐Cre/ERT2 and Lkb1flox/flox;PDGFRα‐Cre/ERT2 mice, performed single‐cell RNA sequencing (scRNA‐seq) and imaging‐based lineage tracing, and aimed to investigate the cellular complexity of gastrointestinal polyps associated with PJS. We found that Lkb1flox/+;Myh11‐Cre/ERT2 mice developed gastrointestinal polyps starting at 9 months after tamoxifen treatment. scRNA‐seq revealed aberrant stem cell‐like characteristics of epithelial cells from polyp tissues of Lkb1flox/+;Myh11‐Cre/ERT2 mice. The Lkb1‐associated polyps were further characterized by a branching smooth muscle core, abundant extracellular matrix deposition, and high immune cell infiltration. In addition, the Spp1–Cd44 or Spp1–Itga8/Itgb1 axes were identified as important interactions among epithelial, mesenchymal, and immune compartments in Lkb1‐associated polyps. These characteristics of gastrointestinal polyps were also demonstrated in another mouse model, tamoxifen‐inducible Lkb1flox/flox;PDGFRα‐Cre/ERT2 mice, which developed obvious gastrointestinal polyps as early as 2–3 months after tamoxifen treatment. Our findings further confirm the critical role of mesenchymal Lkb1/Stk11 in gastrointestinal polyposis and provide novel insight into the cellular complexity of Lkb1‐associated polyp biology. © 2024 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Renin is the key enzyme of the systemic renin–angiotensin–aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin‐1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero‐Cre (P0‐Cre) to abrogate Nrp1 constitutively in P0‐Cre lineage‐labelled cells of the kidney. We found that the P0‐Cre precursor cells differentiate into renin‐producing JG cells. We employed a lineage‐tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell‐autonomous role for NRP1 in JG cell function. Nrp1‐deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0‐Cre–expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. Key points: Renin is a centrepiece of the renin–angiotensin–aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney.Neuropilin‐1 (NRP1) is a conserved membrane‐bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression.We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function.Cell‐specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance.The results support the versatility of renin‐producing cells in the kidney and may open new avenues for therapeutic approaches. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PDE4A–D). Previous studies have shown the beneficial effects of pan-PDE4 inhibitors in rodent PH; however, this class of drugs is associated with side effects owing to the broad inhibition of all four PDE4 isozymes. Here, we demonstrate that PDE4B is the predominant PDE isozyme in lungs and that it was upregulated in rodent and human PH lung tissues. We also confirmed that PDE4B is mainly expressed in the lung endothelial cells (ECs). Evaluation of PH in Pde4b wild type and knockout mice confirmed that Pde4b is important for the vascular remodeling associated with PH. In vivo EC lineage tracing demonstrated that Pde4b induces PH development by driving endothelial-to-mesenchymal transition (EndMT), and mechanistic studies showed that Pde4b regulates EndMT by antagonizing the cAMP-dependent PKA–CREB–BMPRII axis. Finally, treating PH rats with a PDE4B-specific inhibitor validated that PDE4B inhibition has a significant pharmacological effect in the alleviation of PH. Collectively, our findings indicate a critical role for PDE4B in EndMT and PH, prompting further studies of PDE4B-specific inhibitors as a therapeutic strategy for PH. Pan-PDE4 inhibitors attenuate rat pulmonary hypertension (PH), while its applications show serious gastrointestinal side-effects. PDE4B-specific inhibitor may be an advisable therapeutic strategy that can alleviate PH by suppressing EndMT with less side-effects. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single‐cell RNA‐sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R‐GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi‐potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56− quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF‐A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

B-cell repertoires are characterized by a diverse set of receptors of distinct specificities generated through two processes of somatic diversification: V(D)J recombination and somatic hypermutations. B-cell clonal families stem from the same V(D)J recombination event, but differ in their hypermutations. Clonal families identification is key to understanding B-cell repertoire function, evolution, and dynamics. We present HILARy (high-precision inference of lineages in antibody repertoires), an efficient, fast, and precise method to identify clonal families from single- or paired-chain repertoire sequencing datasets. HILARy combines probabilistic models that capture the receptor generation and selection statistics with adapted clustering methods to achieve consistently high inference accuracy. It automatically leverages the phylogenetic signal of shared mutations in difficult repertoire subsets. Exploiting the high sensitivity of the method, we find the statistics of evolutionary properties such as the site frequency spectrum and dN/dS ratio do not depend on the junction length. We also identify a broad range of selection pressures spanning two orders of magnitude.

Organ development, regeneration and cancer initiation are typically influenced by the proliferation and lineage plasticity of tissue-specific stem cells. Prostate intermediate cells, which exhibit characteristics of both basal and luminal cells, are prevalent in pathological states and during organ development. However, the identity, fate and function of these intermediate cells in prostate development are not well understood. Through single-cell RNA-seq analysis on neonatal urogenital sinus tissue, we identified intermediate cells exhibiting stem cell potential. A notable decline in the population of intermediate cells was observed during prostate development. Prostate intermediate cells were specifically labeled in early and late postnatal development by the enhanced dual-recombinase-mediated genetic tracing systems. Our findings revealed that these cells possess significant stem cell capabilities as demonstrated in organoid formation and cell fate mapping assays. These intermediate cells also exhibited intrinsic bipotential properties, enabling them to differentiate into both basal and luminal cells. Additionally, we discovered a novel transition from intermediate cell expressing neuroendocrine markers to neuroendocrine cell during prostate development. This study highlights intermediate cells as a crucial stem cell population and enhances our understanding of their role in prostate development and the plasticity of prostate cancer lineage.

Summary: While most dizygotic twins have a dichorionic placenta, rare cases of dizygotic twins with a monochorionic placenta have been reported. The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified in mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin family to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the β cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet β cells. Cre-LoxP systems were used to generate islet cell type-specific (α, β, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto β-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled β cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of β cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. β cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into β cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of β cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing β cells could also re-trans-differentiate into insulin-producing β cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of β cells. Our findings shed light on how islet β cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis. [ABSTRACT FROM AUTHOR]

Learning how multicellular organs are developed from single cells to different cell types is a fundamental problem in biology. With the high-throughput scRNA-seq technology, computational methods have been developed to reveal the temporal dynamics of single cells from transcriptomic data, from phenomena on cell trajectories to the underlying mechanism that formed the trajectory. There are several distinct families of computational methods including Trajectory Inference (TI), Lineage Tracing (LT), and Gene Regulatory Network (GRN) Inference which are involved in such studies. This review summarizes these computational approaches which use scRNA-seq data to study cell differentiation and cell fate specification as well as the advantages and limitations of different methods. We further discuss how GRNs can potentially affect cell fate decisions and trajectory structures. [ABSTRACT FROM AUTHOR]

Chromosomal instability (CIN), an increased rate of chromosome segregation errors during mitosis, is a hallmark of cancer cells. CIN leads to karyotype differences between cells and thus large-scale heterogeneity among individual cancer cells; therefore, it plays an important role in cancer evolution. Studying CIN and its consequences is technically challenging, but various technologies have been developed to track karyotype dynamics during tumorigenesis, trace clonal lineages and link genomic changes to cancer phenotypes at single-cell resolution. These methods provide valuable insight not only into the role of CIN in cancer progression, but also into cancer cell fitness. In this Cell Science at a Glance article and the accompanying poster, we discuss the relationship between CIN, cancer cell fitness and evolution, and highlight techniques that can be used to study the relationship between these factors. To that end, we explore methods of assessing cancer cell fitness, particularly for chromosomally unstable cancer. [ABSTRACT FROM AUTHOR]

In various biological contexts, cells receive signals and stimuli that prompt them to change their current state, leading to transitions into a future state. This change underlies the processes of development, tissue maintenance, immune response, and the pathogenesis of various diseases. Following the path of cells from their initial identity to their current state reveals how cells adapt to their surroundings and undergo transformations to attain adjusted cellular states. DNA-based molecular barcoding technology enables the documentation of a phylogenetic tree and the deterministic events of cell lineages, providing the mechanisms and timing of cell lineage commitment that can either promote homeostasis or lead to cellular dysregulation. This review comprehensively presents recently emerging molecular recording technologies that utilize CRISPR/Cas systems, base editing, recombination, and innate variable sequences in the genome. Detailing their underlying principles, applications, and constraints paves the way for the lineage tracing of every cell within complex biological systems, encompassing the hidden steps and intermediate states of organism development and disease progression. [ABSTRACT FROM AUTHOR]

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon. [ABSTRACT FROM AUTHOR]

IntroductionHox genes govern rostro-caudal identity along the developing spinal cord, which has a well-defined division of function between dorsal (sensory) and ventral (motor) halves. Here we exploit developmental Hoxb8 expression, normally restricted to the dorsal cord below the obex, to genetically label spinal cord-to-brain (“spinofugal”) axons.MethodsWe crossed two targeted (knock-in) and two non-targeted recombinase-expressing lines (Hoxb8-IRES-Cre and Hoxb8-T2AFlpO; Hoxb8-Cre and Hoxb8-FlpO, respectively) with appropriate tdtomato-expressing reporter strains. Serial sectioning, confocal and superresolution microscopy, as well as light-sheet imaging was used to reveal robust labeling of ascending axons and their terminals in expected and unexpected regions.ResultsThis strategy provides unprecedented anatomical detail of ascending spinal tracts anterior to the brainstem, and reveals a previously undescribed decussating tract in the ventral hypothalamus (the spinofugal hypothalamic decussating tract, or shxt). The absence of Hoxb8-suppressing elements led to multiple instances of ectopic reporter expression in Hoxb8-Cre mice (retinal ganglion and vomeronasal axons, anterior thalamic nuclei and their projections to the anterior cingulate and retrosplenial cortices and subiculum, and a population of astrocytes at the cephalic flexure) and Hoxb8-FlpO mice (Cajal–Retzius cells of the dentate gyrus, and mesenchymal cells of the choroid plexus). While targeted transgenic lines were similar in terms of known spinofugal projections, Hoxb8-IRES-Cre reporters had an additional projection to the core of the facial motor nucleus, and more abundant Hoxb8-lineage microglia scattered throughout the brain than Hoxb8-T2A-FlpO (or any other) mice, suggesting dysregulated Hoxb8-driven reporter expression in one or both lines.DiscussionThis work complements structural and connectivity atlases of the mouse central nervous system, and provides a platform upon which their reactions to injury or disease can be studied. Ectopic Hoxb8-driven recombinase expression may also be a useful tool to study structure and function of other cell populations in non-targeted lines.

Adoptive cell therapy of donor-derived, antigen-specific T cells expressing native T cell receptors (TCRs) is a powerful strategy to fight viral infections in immunocompromised patients. Determining the fate of T cells following patient infusion hinges on the ability to track them in vivo. While this is possible by genetic labeling of parent cells, the applicability of this approach has been limited by the non-specificity of the edited T cells. Here, we devised a method for CRISPR-targeted genome integration of a barcoded gene into Epstein-Barr virus-antigen-stimulated T cells and demonstrated its use for exclusively identifying expanded virus-specific cell lineages. Our method facilitated the enrichment of antigen-specific T cells, which then mediated improved cytotoxicity against Epstein-Barr virus-transformed target cells. Single-cell and deep sequencing for lineage tracing revealed the expansion profile of specific T cell clones and their corresponding gene expression signature. This approach has the potential to enhance the traceability and the monitoring capabilities during immunotherapeutic T cell regimens.

Research in neuroscience has greatly benefited from the development of genetic approaches that enable lineage tracing, cell type targeting, and conditional gene regulation. Recent advances in combinatorial strategies, which integrate multiple cellular features, have significantly enhanced the spatiotemporal precision and flexibility of these manipulations. In this minireview, we introduce the concept and design of these strategies and provide a few examples of their application in genetic fate mapping, cell type targeting, and reversible conditional gene regulation. These advancements have facilitated in‐depth investigation into the developmental principles underlying the assembly of brain circuits, granting experimental access to highly specific cell lineages and subtypes, as well as offering valuable new tools for modeling and studying neurological diseases. Additionally, we discuss future directions aimed at expanding and improving the existing genetic toolkit for a better understanding of the development, structure, and function of healthy and diseased brains. [ABSTRACT FROM AUTHOR]