Cre recombinase microinjection for single-cell tracing and localised gene targeting.

Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.

A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.

An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing ß-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

LACE 2.0: an interactive R tool for the inference and visualization of longitudinal cancer evolution.

BACKGROUND: Longitudinal single-cell sequencing experiments of patient-derived models are increasingly employed to investigate cancer evolution. In this context, robust computational methods are needed to properly exploit the mutational profiles of single cells generated via variant calling, in order to reconstruct the evolutionary history of a tumor and characterize the impact of therapeutic strategies, such as the administration of drugs. To this end, we have recently developed the LACE framework for the Longitudinal Analysis of Cancer Evolution. RESULTS: The LACE 2.0 release aimed at inferring longitudinal clonal trees enhances the original framework with new key functionalities: an improved data management for preprocessing of standard variant calling data, a reworked inference engine, and direct connection to public databases. CONCLUSIONS: All of this is accessible through a new and interactive Shiny R graphical interface offering the possibility to apply filters helpful in discriminating relevant or potential driver mutations, set up inferential parameters, and visualize the results. The software is available at: github.com/BIMIB-DISCo/LACE.

An exciting period of Drosophila developmental biology: Of imaginal discs, clones, compartments, parasegments and homeotic genes.

In this review we recall a number of important discoveries that took place in Drosophila during the seventies and eighties of the last century. The development of cell lineage methods and of powerful modifications of same, such as the Minute technique, led to the discovery of compartments and provided a clearer picture of the body organization: that came to be seen as a chain of metameric lineage units along the A/P body axis. Further, genetic screens allowed the identification of the genes involved in the establishment of the metameric scaffold - the segmentation genes- and also of Hox genes that are responsible for the specific development of individual body parts. As cloning methods became available, many of the most relevant of these developmental genes were cloned and a molecular analysis of development initiated. The discovery of the homeobox, a molecular mark of Hox and other relevant developmental genes, allowed the finding of Hox genes in animal species, like humans, in which they could not be identified by genetic methods. Analysis of the structure and function of Hox genes provided a general image of the genetic design of the metazoan body.

Neural crest lineage analysis: from past to future trajectory.

Since its discovery 150 years ago, the neural crest has intrigued investigators owing to its remarkable developmental potential and extensive migratory ability. Cell lineage analysis has been an essential tool for exploring neural crest cell fate and migration routes. By marking progenitor cells, one can observe their subsequent locations and the cell types into which they differentiate. Here, we review major discoveries in neural crest lineage tracing from a historical perspective. We discuss how advancing technologies have refined lineage-tracing studies, and how clonal analysis can be applied to questions regarding multipotency. We also highlight how effective progenitor cell tracing, when combined with recently developed molecular and imaging tools, such as single-cell transcriptomics, single-molecule fluorescence in situ hybridization and high-resolution imaging, can extend the scope of neural crest lineage studies beyond development to regeneration and cancer initiation.

Fusion transcriptome profiling defines the monoclonal origin of multifocal epithelioid haemangioma of bone.

AIMS: Epithelioid haemangioma (EH) of bone remains a highly controversial entity. Indeed, the WHO classifies EHs of soft tissues as benign tumours, whereas bone EHs are considered intermediate-locally aggressive tumours due to common multifocal presentation and local destructive growth. To gain insights into the clinical behaviour and biology of EH of bone we retrospectively analysed 42 patients treated in a single institution from 1978 to 2021. METHODS AND RESULTS: Multifocal presentation was detected in 17 of 42 patients (40%) primarily as synchronous lesions. Patients were treated with curettage (57%), resection (29%) or biopsy, followed by radiotherapy or embolisation (14%). Follow-up (minimum 24 months) was available for 38 patients, with only five local recurrences (13%) and no death of disease. To clarify whether the synchronous bone lesions in multifocal EH represent multicentric disease or clonal dissemination, four cases were profiled by RNA-sequencing. Separate lesions from the same patient, which showed a similar transcriptional profile, expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints. CONCLUSIONS: These results indicate that, in EH of bone, multifocal lesions are clonally related and therefore represent the spread of a same neoplastic clone rather than simultaneous independent tumours. This finding is in apparent contradiction with the benign clinical course of the disease, and suggests that tumour dissemination in bone EH probably reflects a phenomenon of passive spreading, with tumour cells colonising distal sites while maintaining their benign biological nature.

Epidemiology of invasive Haemophilus influenzae disease in northwestern Ontario: comparison of invasive and noninvasive H. influenzae clinical isolates.

In the post-Haemophilus influenzae type b (Hib) vaccine era, invasive H. influenzae type a (Hia) disease emerged in North American Indigenous populations. The role of Hia in noninvasive disease is uncertain; it is unknown whether noninvasive Hia infections are prevalent in populations with a high incidence of invasive disease, and whether invasive and noninvasive Hia isolates have different characteristics. We analyzed all invasive and noninvasive clinical H. influenzae isolates collected in a northwestern Ontario hospital serving 82% Indigenous population over 5.5 years. Serotyping, clonal analysis, and antimicrobial sensitivity testing were conducted on 233 noninvasive and 20 invasive isolates. Among noninvasive isolates, 91% were nontypeable (NTHi) and 3% were Hia; Hia was the most frequent invasive isolate (60%). Incidence rates of invasive H. influenzae disease (12.5/100 000/year) greatly exceeded average provincial data, with the highest found in <6-year-old children (63.9/100 000/year); the proportion of Hia among invasive isolates was seven times larger than in Ontario. No difference in clonal characteristics between invasive and noninvasive Hia isolates was found. Antibiotic resistance was more common among NTHi than among encapsulated isolates, without differences between invasive and noninvasive isolates. Considering the significance of Hia in Indigenous populations, pediatric immunization against Hia will be useful to prevent serious infections in young Indigenous children.

From one cell to many: Morphogenetic field of lateral root founder cells in Arabidopsis thaliana is built by gradual recruitment.

The reiterative process of lateral root (LR) formation is widespread and underlies root system formation. However, early LR primordium (LRP) morphogenesis is not fully understood. In this study, we conducted both a clonal analysis and time-lapse experiments to decipher the pattern and sequence of pericycle founder cell (FC) participation in LR formation. Most commonly, LRP initiation starts with the specification of just one FC longitudinally. Clonal and anatomical analyses suggested that a single FC gradually recruits neighboring pericycle cells to become FCs. This conclusion was validated by long-term time-lapse live-imaging experiments. Once the first FC starts to divide, its immediate neighbors, both lengthwise and laterally, are recruited within the hour, after which they recruit their neighboring cells within a few hours. Therefore, LRP initiation is a gradual, multistep process. FC recruitment is auxin-dependent and is abolished by treatment with a polar auxin transport inhibitor. Furthermore, FC recruitment establishes a morphogenetic field where laterally peripheral cells have a lower auxin response, which is associated with a lower proliferation potential, compared to centrally located FCs. The lateral boundaries of the morphogenetic field are determined by phloem-adjacent pericycle cells, which are the last cells to be recruited as FCs. The proliferation potential of these cells is limited, but their recruitment is essential for root system formation, resulting in the formation of a new vascular connection between the nascent and parent root, which is crucial for establishing a continuous and efficient vascular system.

Clonal analysis by tunable CRISPR-mediated excision.

Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here, we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum We demonstrate that clone marking frequency can be tuned to generate embryos that carry single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.

Deciphering neural heterogeneity through cell lineage tracing.

Understanding how an adult brain reaches an appropriate size and cell composition from a pool of progenitors that proliferates and differentiates is a key question in Developmental Neurobiology. Not only the control of final size but also, the proper arrangement of cells of different embryonic origins is fundamental in this process. Each neural progenitor has to produce a precise number of sibling cells that establish clones, and all these clones will come together to form the functional adult nervous system. Lineage cell tracing is a complex and challenging process that aims to reconstruct the offspring that arise from a single progenitor cell. This tracing can be achieved through strategies based on genetically modified organisms, using either genetic tracers, transfected viral vectors or DNA constructs, and even single-cell sequencing. Combining different reporter proteins and the use of transgenic mice revolutionized clonal analysis more than a decade ago and now, the availability of novel genome editing tools and single-cell sequencing techniques has vastly improved the capacity of lineage tracing to decipher progenitor potential. This review brings together the strategies used to study cell lineages in the brain and the role they have played in our understanding of the functional clonal relationships among neural cells. In addition, future perspectives regarding the study of cell heterogeneity and the ontogeny of different cell lineages will also be addressed.

Single Cell Approaches to Understand the Earliest Steps in Heart Development.

PURPOSE OF REVIEW: Cardiac progenitors are the building blocks of the heart. Our knowledge, on how these progenitors build the heart, has considerably increased over the last two decades with the development of single cell approaches. We discuss the lessons learnt from clonal analyses and from single cell sequencing technologies on the understanding of the earliest steps of cardiac specification and lineage segregation. RECENT FINDINGS: While experiments were initially performed at the population level, the development of approaches to investigate heart development at the single cell resolution has clearly demonstrated that cardiac progenitors are highly heterogeneous, with different progenitors contributing to different cardiac regions and different cardiac cell types. Some critical transcriptional determinants have also been identified for cardiac progenitor specification. Single cell approaches have finally provided insights into the spatio-temporal specification of unipotent and multipotent cardiac progenitors and provided a framework for investigating congenital heart defects.

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis.

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells.

[Clonal analysis of hematopoietic stem cells].

Hematopoietic stem cells (HSCs) are defined as cells present in the bone marrow that are capable of both self-renewal and multilineage differentiation. The differentiation pathway from HSCs to mature blood cells and HSC self-renewal and differentiation mechanisms still remain unclear and require further elucidation. However, HSCs are a highly diverse population with analysis limitations at the population level. Moreover, molecular and cell biological single-cell analysis has been attracting tremendous attention recently. Herein, we introduce a cell biological single-cell analysis method (single-cell transplantation and cell lineage tracing experiments using genetic techniques) in the field of HSCs.

Amniotic ectoderm expansion in mouse occurs via distinct modes and requires SMAD5-mediated signalling.

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis.

Recent advances in single-cell multimodal analysis to study immune cells.

Recent advances in single-cell technologies have enabled the profiling of the genome, epigenome, transcriptome and proteome, along with temporal and spatial information of individual cells. These technologies have provided unique opportunities to understand mechanisms underpinning the immune system, such as characterizations of the molecular cell state, how the cell state evolves along its lineage and the impact of spatial location on cell state. In this review, we discuss how these mechanisms have been studied through recent advances in single-cell multimodal technologies.

Life-Long Neural Stem Cells Are Fate-Specified at an Early Developmental Stage.

The origin and life-long fate of quiescent neural stem cells (NSCs) in the adult mammalian brain remain largely unknown. A few neural precursor cells in the embryonic brain elongate their cell cycle time and subsequently become quiescent postnatally, suggesting the possibility that life-long NSCs are selected at an early embryonic stage. Here, we utilized a GFP-expressing lentivirus to investigate the fate of progeny from individual lentivirus-infected NSCs by identifying the lentiviral integration site. Our data suggest that NSCs become specified to two or more lineages prior to embryonic day 13.5 in mice: one NSC lineage produces cells only for the cortex and another provides neurons to the olfactory bulb. The majority of neurosphere-forming NSCs in the adult brain are relatively dormant and generate very few cells, if any, in the olfactory bulb or cortex, and this NSC population could serve as a reservoir that is occasionally reactivated later in life.

Multiplex clonal analysis in the chick embryo using retrovirally-mediated combinatorial labeling.

Lineage analysis plays a central role in exploring the developmental potential of stem and progenitor cell populations. In higher vertebrates, a variety of techniques have been used to label individual cells or cell populations, including interspecies grafting, intracellular microinjection, and Cre-mediated recombination. However, these approaches often suffer from difficulties in progenitor cell targeting, low cellular resolution and/or ectopic labeling. To circumvent these issues, here we utilize replication incompetent avian (RIA) retroviruses to deliver combinations of fluorescent proteins into distinct cellular compartments in chick embryos. In particular, RIA-mediated lineage tracing is optimal for long term mapping of dispersing cell populations like the neural crest. Using this tool, we confirm that trunk neural crest cells are multipotent. Furthermore, our RIA vector is engineered to be fully adaptable for other purposes such as cell fate analysis, gene perturbation studies and time-lapse imaging. Taken together, we present a novel approach of multiplex lineage analysis that can be applied to normal and perturbed development of diverse cell populations in avian embryos.

Live fate-mapping of joint-associated fibroblasts visualizes expansion of cell contributions during zebrafish fin regeneration.

The blastema is a mass of progenitor cells responsible for regeneration of amputated salamander limbs and fish fins. Previous studies have indicated that resident cell sources producing the blastema contribute lineage-restricted progeny to regenerating tissue. However, these studies have labeled general cell types rather than granular cell subpopulations, and they do not explain the developmental transitions that must occur for distal structures to arise from cells with proximal identities in the appendage stump. Here, we find that regulatory sequences of tph1b, which encodes an enzyme that synthesizes serotonin, mark a subpopulation of fibroblast-like cells restricted to the joints of uninjured adult zebrafish fins. Amputation stimulates serotonin production in regenerating fin fibroblasts, yet targeted tph1b mutations abrogating this response do not disrupt fin regeneration. In uninjured animals, tph1b-expressing cells contribute fibroblast progeny that remain restricted to joints throughout life. By contrast, upon amputation, tph1b+ joint cells give rise to fibroblasts that distribute across the entire lengths of regenerating fin rays. Our experiments visualize and quantify how incorporation into an appendage blastema broadens the progeny contributions of a cellular subpopulation that normally has proximodistal restrictions.

A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury.

Limbal epithelial stem cells are required for the maintenance and repair of the corneal epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. Porcine corneal tissue represents an attractive experimental model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of this tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed that in response to central epithelial wounding, a greater proportion of progenitors in the basal limbal epithelium enter an actively dividing state. The authors present a comprehensively validated model system for studying the interactions between limbal niche factors and limbal epithelial stem cell fate.

Positional cues regulate dorsal organ formation in the liverwort Marchantia polymorpha.

Bryophytes and vascular plants represent the broadest evolutionary divergence in the land plant lineage, and comparative analyses of development spanning this divergence therefore offer opportunities to identify truisms of plant development in general. In vascular plants, organs are formed repetitively around meristems at the growing tips in response to positional cues. In contrast, leaf formation in mosses and leafy liverworts occurs from clonal groups of cells derived from a daughter cell of the apical stem cell known as merophytes, and cell lineage is a crucial factor in repetitive organ formation. However, it remains unclear whether merophyte lineages are a general feature of repetitive organ formation in bryophytes as patterns of organogenesis in thalloid liverworts are unclear. To address this question, we developed a clonal analysis method for use in the thalloid liverwort Marchantia polymorpha, involving random low-frequency induction of a constitutively expressed nuclear-targeted fluorescent protein by dual heat-shock and dexamethasone treatment. M. polymorpha thalli ultimately derive from stem cells in the apical notch, and the lobes predominantly develop from merophytes cleft to the left and right of the apical cell(s). Sector induction in gemmae and subsequent culture occasionally generated fluorescent sectors that bisected thalli along the midrib and were maintained through several bifurcation events, likely reflecting the border between lateral merophytes. Such thallus-bisecting sectors traversed dorsal air chambers and gemma cups, suggesting that these organs arise independently of merophyte cell lineages in response to local positional cues.