Lineage Tracing Techniques Research Articles

A stress-induced miR-31-CLOCK-ERK pathway is a key driver and therapeutic target for skin aging.

Regressive changes in epithelial stem cells underlie mammalian skin aging, but the driving mechanisms are not well understood. Here, we report that mouse skin hair follicle stem cell (HFSC) aging is initiated by their intrinsic upregulation of miR-31, a microRNA that can be induced by physical injury or genotoxic stress and is also strongly upregulated in aged human skin epithelium. Using transgenic and conditional knockout mouse models plus a lineage-tracing technique, we show that miR-31 acts as a key driver of HFSC aging by directly targeting Clock, a core circadian clock gene whose deregulation activates a MAPK/ERK cascade to induce HFSC depletion via transepidermal elimination. Notably, blocking this pathway by either conditional miR-31 ablation or clinically approved MAPK/ERK inhibitors provides safe and effective protection against skin aging, enlightening a promising therapeutic avenue for treating skin aging and other genotoxic stress-induced skin conditions such as radiodermatitis.

Recent advances in lineage tracing for the kidney

Lineage tracing was originally developed by developmental biologists to identify all progeny of a single cell during morphogenesis. More recently this approach has been applied to other fields, including organ homeostasis and recovery from injury. Modern lineage tracing techniques typically rely on reporter gene expression induced by cell-specific DNA recombination. There have been important scientific advances in the last 10 years that have impacted lineage tracing approaches, including intersectional genetics, optical clearing techniques, and the use of sequencing-based genomic lineage tracing. The latter combines CRISPR-Cas9-based genetic scarring with single-cell RNA-sequencing that, in theory, could allow comprehensive reconstruction of a lineage tree for an entire organism. This review summarizes recent advances in lineage tracing technologies and outlines potential applications for kidney research.

The Plasticity of Pancreatic β-Cells

Type 2 diabetes is caused by impaired insulin secretion and/or insulin resistance. Loss of pancreatic β-cell mass detected in human diabetic patients has been considered to be a major cause of impaired insulin secretion. Additionally, apoptosis is found in pancreatic β-cells; β-cell mass loss is induced when cell death exceeds proliferation. Recently, however, β-cell dedifferentiation to pancreatic endocrine progenitor cells and β-cell transdifferentiation to α-cell was reported in human islets, which led to a new underlying molecular mechanism. Hyperglycemia inhibits nuclear translocation and expression of forkhead box-O1 (FoxO1) and induces the expression of neurogenin-3 (Ngn3), which is required for the development and maintenance of pancreatic endocrine progenitor cells. This new hypothesis (Foxology) is attracting attention because it explains molecular mechanism(s) underlying β-cell plasticity. The lineage tracing technique revealed that the contribution of dedifferentiation is higher than that of β-cell apoptosis retaining to β-cell mass loss. In addition, islet cells transdifferentiate each other, such as transdifferentiation of pancreatic β-cell to α-cell and vice versa. Islet cells can exhibit plasticity, and they may have the ability to redifferentiate into any cell type. This review describes recent findings in the dedifferentiation and transdifferentiation of β-cells. We outline novel treatment(s) for diabetes targeting islet cell plasticity.

From Stripes to a Beating Heart: Early Cardiac Development in Zebrafish.

The heart is the first functional organ to form during vertebrate development. Congenital heart defects are the most common type of human birth defect, many originating as anomalies in early heart development. The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease. Although composed of only two chambers compared with the four-chambered mammalian heart, the zebrafish heart integrates the core processes and cellular lineages central to cardiac development across vertebrates. The rapid, translucent development of zebrafish is amenable to in vivo imaging and genetic lineage tracing techniques, providing versatile tools to study heart field migration and myocardial progenitor addition and differentiation. Combining transgenic reporters with rapid genome engineering via CRISPR-Cas9 allows for functional testing of candidate genes associated with congenital heart defects and the discovery of molecular causes leading to observed phenotypes. Here, we summarize key insights gained through zebrafish studies into the early patterning of uncommitted lateral plate mesoderm into cardiac progenitors and their regulation. We review the central genetic mechanisms, available tools, and approaches for modeling congenital heart anomalies in the zebrafish as a representative vertebrate model.

Research progress in the identification and lineage differentiation of skeletal stem cell

Skeletal system contains various stem cell niches in which the self-renewal and differentiation of different kinds of stem cells are supported. Stem cell-based therapy has been applicated variedly. The skeletal stem cells (SSC), which are mesenchyme-derived and skeletal-lineage committed progenitors, can be found in the skeletal system. They are able to self-renew and colone both in vivo and in vitro. SSC have the potential of multi-directional differentiation to cartilage, bone, adipocytes and marrow stroma. Several types of SSC have been identified based on lineage tracing and single-cell RNA sequencing technique. Compared to mesenchymal stem cells, SSC are groups of less heterogeneous cells with accurate surface marker and identifiable biological behavior. Thus, clarifying the properties of SSC might provide new insight for stem cell therapy. In this article, the authors retrieved and reviewed SSC researches mainly focusing on the identification, spatio-temporal distribution and functional characteristics of SSC. The application of scRNA-seq in skeletal stem cell identification and functional study was also discussed.

Osteoblast Lineage Support of Hematopoiesis in Health and Disease.

In mammals, hematopoiesis migrates to the bone marrow during embryogenesis coincident with the appearance of mineralized bone, where hematopoietic stem cells (HSCs) and their progeny are maintained by the surrounding microenvironment or niche, and sustain the entirety of the hematopoietic system. Genetic manipulation of niche factors and advances in cell lineage tracing techniques have implicated cells of both hematopoietic and nonhematopoietic origin as important regulators of hematopoiesis in health and disease. Among them, cells of the osteoblast lineage, from stromal skeletal stem cells to matrix-embedded osteocytes, are vital niche residents with varying capacities for hematopoietic support depending on stage of differentiation. Here, we review populations of osteoblasts at differing stages of differentiation and summarize the current understanding of the role of the osteoblast lineage in supporting hematopoiesis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Looking at the developing lung in single-cell resolution.

Lung development is a complicated and delicate process, facilitated by spatially and temporarily coordinated cross talk of up to 40 cell types. Developmental origin and heterogeneity of lung cell lineages in context of lung development have been a focus of research efforts for decades. Bulk RNA and protein measurements, RNA and protein labeling, and lineage tracing techniques have been traditionally employed. However, the complex and heterogeneous nature of lung tissue presents a particular challenge when identifying subtle changes in gene expression in individual cell types. Rapidly developing single-cell RNA sequencing (scRNA-seq) techniques allow for unbiased and robust assessment of complex cellular dynamics during biological processes in unprecedented ways. Discovered a decade ago, scRNA-seq has been applied in respiratory research to understand lung cellular composition and to identify novel cell types. Still, very few studies to date have addressed the single-cell transcriptome in healthy or aberrantly developing lung. In this review, we discuss principal discoveries with scRNA-seq in the field of prenatal and postnatal lung development. In addition, we examine challenges and expectations, and propose future steps associated with the use of scRNA-seq to study developmental lung diseases.

Markers for Identification of Postnatal Skeletal Stem Cells In Vivo.

The adult skeleton contains stem cells involved in growth, homeostasis, and healing. Mesenchymal or skeletal stem cells are proposed to provide precursors to osteoblasts, chondrocytes, marrow adipocytes, and stromal cells. We review the evidence for existence and functionality of different skeletal stem cell pools, and the tools available for identifying or targeting these populations in mouse and human tissues. Lineage tracing and single cell-based techniques in mouse models indicate that multiple pools of stem cells exist in postnatal bone. These include growth plate stem cells, stem and progenitor cells in the diaphysis, reticular cells that only form bone in response to injury, and injury-responsive periosteal stem cells. New staining protocols have also been described for prospective isolation of human skeletal stem cells. Several populations of postnatal skeletal stem and progenitor cells have been identified in mice, and we have an increasing array of tools to target these cells. Most Cre models lack a high degree of specificity to define single populations. Human studies are less advanced and require further efforts to refine methods for identifying stem and progenitor cells in adult bone.

Impact of Local Alloimmunity and Recipient Cells in Transplant Arteriosclerosis.

Transplant arteriosclerosis is the major limitation to long-term survival of solid organ transplantation. Although both immune and nonimmune cells have been suggested to contribute to this process, the complex cellular heterogeneity within the grafts, and the underlying mechanisms regulating the disease progression remain largely uncharacterized. We aimed to delineate the cellular heterogeneity within the allografts, and to explore possible mechanisms underlying this process. Here, we reported the transcriptional profiling of 11 868 cells in a mouse model of transplant arteriosclerosis by single-cell RNA sequencing. Unbiased clustering analyses identified 21 cell clusters at different stages of diseases, and focused analysis revealed several previously unknown subpopulations enriched in the allografts. Interestingly, we found evidence of the local formation of tertiary lymphoid tissues and suggested a possible local modulation of alloimmune responses within the grafts. Intercellular communication analyses uncovered a potential role of several ligands and receptors, including Ccl21a and Cxcr3, in regulating lymphatic endothelial cell-induced early chemotaxis and infiltration of immune cells. In vivo mouse experiments confirmed the therapeutic potential of CCL21 and CXCR3 neutralizing antibodies in transplant arteriosclerosis. Combinational use of genetic lineage tracing and single-cell techniques further indicate the infiltration of host-derived c-Kit+ stem cells as heterogeneous populations in the allografts. Finally, we compared the immune response between mouse allograft and atherosclerosis models in single-cell RNA-seq analysis. By analyzing susceptibility genes of disease traits, we also identified several cell clusters expressing genes associated with disease risk. Our study provides a transcriptional and cellular landscape of transplant arteriosclerosis, which could be fundamental to understanding the initiation and progression of this disease. CCL21/CXCR3 was also identified as important regulators of immune response and may serve as potential therapeutic targets in disease treatment.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Spotlight on Tendon: Its Novel Role in Long Bone Growth and Rickets‐Caused Osteosclerosis

INTRODUCTIONIt has been known for many years that tendon‐attached bone is rough and bump, and that patients with hypophosphatemic rickets (such as DMP1 mutations) develop osteosclerosis in the tendon‐attached area. The goal of this study was to test whether tendon directly forms bone and how hypophosphatemia initiates the onset of osteosclerosis in a Dmp1 KO mouse (a hypophosphatemic rickets model) using cell lineage tracing and multiple imaging techniques.METHODSWe generated a chondrocyte tracing mouse line (Col 10a1‐Cre;R26R tomato) and a tendon tracing mouse line (Scx‐CreERT2;R26R tomato) with and without a Dmp1 KO background, plus a high Pi rescue diet treatment for seven weeks.RESULTSOur key findings were: 1) the non‐tendon attached bone cells were negative in both tracing lines; 2) cells in the tendon‐attached bone were Scx+ but Col X−; 3). In the Dmp1 KO mice, tendon‐attached bone volume sharply increased, in which there were more EdU+ bone cells (i.e., more cell proliferation) with high expression levels of bone markers such as Runx2 and β‐catenin; and 4) a high Pi diet greatly improved the above phenotypes.CONCLUSIONTendon (an avascular tissue) directly forms bone cells in the tendon‐bone joint area, which is rough and porous compared to non‐tendon attached bones. Osteosclerosis in Dmp1 KO (a hypophosphatemia rickets model) is caused by accelerated cell transdifferentiation from tendon to bone due to hypophosphatemia.Support or Funding InformationU.S. National Institutes of Health grants to J.Q.F. (R01DE025014 and DE025659)

Defining Adult Stem Cell Function at Its Simplest: The Ability to Replace Lost Cells through Mitosis.

Classic studies on hematopoiesis indicate that blood cell numbers are maintained by rare, hard-wired, transplantable stem cells (SCs). Subsequent studies in other organs have implicitly assumed that all SC hierarchies follow the design of the hematopoietic system. Lineage tracing techniques have revolutionized the study of solid tissue SCs. It thus appears that key characteristics of the hematopoietic SC hierarchy (rarity of SCs, specific marker expression, quiescence, asymmetric division, and unidirectional differentiation) are not generalizable to other tissues. In light of these insights, we offer a revised, generalizable definition of SC function: the ability to replace lost tissue through cell division.

Biological Characteristics and Regulation of Early Megakaryocytopoiesis.

For decades, megakaryocytopoiesis is believed to occur following a classical binary hierarchical developmental model. This model is based on an analysis of predefined flow-sorted cell populations by using cell surface markers. However, this classical model has been challenged by increasing evidences obtained with new techniques which integrating flow cytometric, transcriptomic and functional data at single-cell level and with lineage tracing technique. These recent advances in megakaryocytopoiesis proposed that commitment of haematopoietic stem cells (HSCs) towards megakaryocytic lineage occurs in much earlier stage than that postulated in the classical model. There may exist multipotent but megakaryocyte (MK)/platelet-biased HSCs within HSC compartment and even HSCs can directly differentiate into MKs in steady state or in response to stress. In this review, we focus on recent findings about differentiation from commitment of HSCs to MK and its regulation, and discuss future directions in this research field.

Mesencephalic origin of the inferior lobe in zebrafish

BackgroundAlthough the overall brain organization is shared in vertebrates, there are significant differences within subregions among different groups, notably between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish). Recent comparative studies focusing on the ventricular morphology have revealed a large diversity of the hypothalamus. Here, we study the development of the inferior lobe (IL), a prominent structure forming a bump on the ventral surface of the teleost brain. Based on its position, IL has been thought to be part of the hypothalamus (therefore forebrain).ResultsTaking advantage of genetic lineage-tracing techniques in zebrafish, we reveal that cells originating from her5-expressing progenitors in the midbrain-hindbrain boundary (MHB) participate in the formation of a large part of the IL. 3D visualization demonstrated how IL develops in relation to the ventricular system. We found that IL is constituted by two developmental components: the periventricular zone of hypothalamic origin and the external zone of mesencephalic origin. The mesencephalic external zone grows progressively until adulthood by adding new cells throughout development.ConclusionOur results disprove a homology between the IL and the mammalian lateral hypothalamus. We suggest that the IL is likely to be involved in multimodal sensory integration rather than feeding motivation. The teleost brain is not a simpler version of the mammalian brain, and our study highlights the evolutionary plasticity of the brain which gives rise to novel structures.

Developmental analyses of mouse embryos and adults using a non-overlapping tracing system for all three germ layers.

Genetic lineage-tracing techniques are powerful tools for studying specific cell populations in development and pathogenesis. Previous techniques have mainly involved systems for tracing a single gene, which are limited in their ability to facilitate direct comparisons of the contributions of different cell lineages. We have developed a new combinatorial system for tracing all three germ layers using self-cleaving 2A peptides and multiple site-specific recombinases (SSRs). In the resulting TRiCK (TRiple Coloured germ layer Knock-in) mice, the three germ layers are conditionally and simultaneously labelled with distinct fluorescent proteins via embryogenesis. We show that previously reported ectopic expressions of lineage markers are the outcome of secondary gene expression. The results presented here also indicate that the commitment of caudal axial stem cells to neural or mesodermal fate proceeds without lineage fluctuations, contrary to the notion of their bi-potency. Moreover, we developed IMES, an optimized tissue clearing method that is highly compatible with a variety of fluorescent proteins and immunostaining, and the combined use of TRiCK mice and IMES can facilitate comprehensive analyses of dynamic contributions of all three germ layers.

Application of Cell Lineage Tracing Combined with Immunofluorescence in the Study of Dentinogenesis.

The cell lineage tracing system has been used predominantly in developmental biology studies. The Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. In this protocol, we will introduce how the cell lineage tracing technique can be performed in the investigation of dentinogenesis by using Gli1-CreERT2; R26RTomato compound mice. Moreover, we combined cell lineage tracing in conjunction with immunofluorescence-to further define cell fate by analyzing the expression of specific cell markers for odontoblasts. This combination not only broadens the application of cell lineage tracing but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation status of parent cell progeny can be displayed simultaneously, providing more information than cell lineage tracing or immunofluorescence alone. In conclusion, the co-application of cell lineage tracing technique and immunofluorescence is a powerful tool for investigating cell biology in the field of dentinogenesis and tooth development.

Critical Roles of Endochondral Bone Formation in Mandible Trauma Repair

Regeneration of bony defects in the mandible remains a significant challenge in oral and maxillofacial surgery. The role of the membranous bone response in bony regeneration of the mandible is well-understood. However, recent studies have shown that cartilage grafts can also lead to regeneration of bone as effectively as autogenous bone grafts, and better than allografts. Although the mechanism is largely unknown, a direct transformation from chondrocytes into bone cells in the mandible during bone development, growth, and repair has been demonstrated1,2. In this study, we investigate the mechanism of endochondral bone formation via the direct transformation of chondrocytes into bone forming cells during mandibular body and symphyseal trauma repair using cell lineage tracing techniques.

Guidelines and best practices in successfully using Zebrabow for lineage tracing multiple cells within tissues.

Labelling cells and following their progeny, also known as lineage tracing, has provided important insights into the cellular origins of tissues. Traditional lineage tracing experiments have been limited to following single or small groups of cells with classic techniques such as dye injections and Cre/LoxP labelling of cells of interest. Brainbow is a fluorescent dependent, lineage tracing technique that allows a broader visualization and analysis of multiple cells within a tissue, initially deployed to examine lineages within neural tissues. This technique has now been adapted to zebrafish (Zebrabow) and takes advantages of the imaging capabilities that this system provides over other animal models. In this paper we shall describe how Zebrabow is performed as well as some guides on some of the common pitfalls encountered when using this labelling strategy.

Abstract 3301: Using a novel single-cell lineage-tracing technique to uncover the mechanisms driving nongenetic cancer relapse

Abstract Despite favorable initial response to platinum chemotherapy, the majority of patients with ovarian cancer will develop recurrent disease and succumb to it within 5 years of diagnosis. Initial recurrence is frequently platinum-sensitive and women can respond to multiple lines of platinum-based chemotherapy before eventually developing platinum-refractory disease. The observation that the tumor cells that survive the initial therapy give rise to a chemosensitive progeny is inconsistent with a simple Darwinian model of selection of genetically resistant clones. Tumor dormancy, stochastic cell state shifts and stem cell-like populations are among the mechanisms hypothesized to mediate re-emergence of platinum-sensitive ovarian cancer. However, given the lack of high-throughput methods to simultaneously track cell state and lineage, it is not currently feasible to distinguish the relative contribution of each of these factors. To address this need, we generated a new lentiviral barcode library, termed Watermelon. Unlike existing barcoding libraries that enable the tracking of multiple cancer cells solely at the DNA level, the Watermelon library encodes transcribed barcodes, which allows one to map the lineage to the transcriptional profile of each individual cell. This unique barcoding scheme, which is coupled with a genetic H2B retention system, enables simultaneous tracing of lineage as well as the transcriptional and proliferative states of each cell in the population during drug treatment. We have established and characterized an in vitro platinum-sensitive relapse model and are applying the Watermelon system together with time-lapse imaging to study the mechanisms underlying time-to-relapse variation and cell-fate decisions. By broadening our knowledge of the cellular and molecular pathways that affect noninherited drug resistance, our work will facilitate the future development of novel therapies that delay or even prevent the emergence of platinum-refractory ovarian cancer. Citation Format: Yaara Oren, Aviv Regev, Joan Brugge. Using a novel single-cell lineage-tracing technique to uncover the mechanisms driving nongenetic cancer relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3301.

Pax4 Gene Transfer Induces α-to-ß Transdifferentiation and Improves the Therapeutic Efficacy of Islet Transplantation

Pax4 is a transcriptional factor which plays a critical role in the generation of insulin producing β-cells during embryonic development. Our group previously demonstrated that Pax4 delivered by adenoviral vector induces insulin expression and reduces glucagon in αTC1.9 cells and primary human islets. The purpose of the present study was to determine whether Pax4 gene expression induces pancreatic α-cells to become β-cell using lineage tracing techniques and confers therapeutic benefits in the context of islet transplantation. In this study, we generated α mouse model in which their α cells are labelled with YFP, namely αYFP mouse. Using this mouse model, we found that Ad5.Pax4 induced glucagon secreting α-cells to become insulin secreting β-cells in isolated αYFP islets. Quantification of YFP+/Pax4+/insulin+ cells confirmed that Pax4 induced α-to-β cell conversion as assessed by the presence of YFP reporter in new β-cells. In addition, these Pax4-treated islets increased glucose-stimulated insulin secretion compare to control group. Furthermore, we transplanted Pax4-treated primary mouse islets into kidney capsule of the streptozotocin-induced diabetic mice and found that they significantly reduced the blood glucose and improved glucose tolerance compared to control groups. These data indicate that Pax4 induced α- to functional β-cell conversion, and proved therapeutic benefits for islet transplantation. Disclosure K.R. Parajuli: None. Y. Zhang: None. H. Wu: None.