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Early childhood malignancies are driven by sparse genetic aberrations in oncogenes that often co-occur with large copy number variants (CNVs). The combination of these mutations is thought to transform developmentally pliant embryonic cells to initiate tumorigenesis. However, the mechanistic interactions between CNVs, oncogenes, and differentiation have not been systematically studied due to several obstacles: (i) CNVs cannot be engineered efficiently yet; (ii) transient embryonic progenitors are absent in full-grown tumors; and (iii) inter-species differences in lineage specification limit the applicability of animal models. To overcome these challenges, we used isogenic human embryonic stem cell (hESC) lines carrying gains of chromosome 17q/1q, which are prevalent in the embryonal tumor neuroblastoma (NB). We differentiated these cells toward trunk neural crest (NC) and their sympathoadrenal derivatives, the putative cells-of-origin of NB, and performed single-cell RNA sequencing and cell-biological assays at key differentiation stages. We found that CNVs impaired the specification of sympathoadrenal cell types and instead potentiated early Schwann-cell-precursor-like phenotypes. Additional overexpression of the oncogene MYCN (which is frequently amplified together with CNVs in high-risk NB tumors) exacerbated these differentiation defects, enabled tumourigenic cell proliferation, and generated cell states in vitro that transcriptionally resembled NB tumor cells. Finally, using epigenome analysis, we connected these states to a stepwise disruption of gene-regulatory networks centered on developmental transcription factors. Together, our results chart a mechanistic route to NB tumorigenesis and provide a general framework for the CNV-driven initiation of embryonal tumors, in which CNVs ‘prime’ embryonic cells for oncogenic transformation. The tumor-like cells in our model may serve as proxies to experimentally test therapeutic interventions during tumorigenesis. Citation Format: Ingrid M. Saldana-Guerrero, Luis F. Montano-Gutierrez, Christoph Hafemeister, Dylan Stavish, Lisa E. Shaw, Irfete S. Fetahu, Andrea Wenninger-Weinzierl, Caterina Sturtzel, Celine Souilhol, Sophia Tarelli, Mohamed R. Shoeb, Marie Bernkopf, Polyxeni Bozatzi, Maria Guarini, Eva Bozsaky, Michelle C. Buri, Eva M. Putz, Peter W. Andrews, Ivana Barbaric, Helen E. Bryant, Martin Distel, Sabine Taschner-Mandl, Matthias Farlik, Anestis Tsakiridis, Florian Halbritter. A stem cell model dissects detrimental effects of neuroblastoma-linked chromosomal aberrations on cell differentiation during neural crest development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3542.

Summary The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS., Highlights • Retinoic acid alters the axial identity of hPSC-derived neural crest cells • ENS progenitor markers are upregulated in response to RA • ENS progenitors are capable of generating enteric neurons in vitro • hPSC ENS progenitors colonize the ENS of mice following long-term transplantation, In this article, Frith and colleagues show that retinoic acid (RA) signaling alters the axial identity of hPSC-derived neural crest cells in a time- and dose-dependent manner. They utilized this to derive enteric nervous system (ENS) progenitors from hSPCs, which can differentiate to enteric neurons in vitro and colonize the ENS of adult mice following long-term transplantation.

The production of the tissues that make up the mammalian embryonic trunk takes place in a head-tail direction, via the differentiation of posteriorly-located axial progenitor populations. These include bipotent neuromesodermal progenitors (NMPs), which generate both spinal cord neurectoderm and presomitic mesoderm, the precursor of the musculoskeleton. Over the past few years, a number of studies have described the derivation of NMP-like cells from mouse and human pluripotent stem cells (PSCs). In turn, these have greatly facilitated the establishment of PSC differentiation protocols aiming to give rise efficiently to posterior mesodermal and neural cell types, which have been particularly challenging to produce using previous approaches. Moreover, the advent of 3-dimensional-based culture systems incorporating distinct axial progenitor-derived cell lineages has opened new avenues toward the functional dissection of early patterning events and cell vs non-cell autonomous effects. Here, we provide a brief overview of the applications of these cell types in disease modelling and cell therapy and speculate on their potential uses in the future.

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of the childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body (NMPs). Here we employ human embryonic stem cell differentiation to define how NMP-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. In contrast, posteriorisation of NMP-derived spinal cord cells is TBXT/WNT-independent and takes place under the influence of FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron-based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)+ spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Differentiation of neuromesodermal progenitors Basic Protocol 2: Posterior ventral spinal cord progenitor differentiation Basic Protocol 3: Posterior motor neuron differentiation.

The intrinsic innervation of the gastrointestinal (GI) tract is comprised of enteric neurons and glia, which are buried within the wall of the bowel and organized into two concentric plexuses that run along the length of the gut forming the enteric nervous system (ENS). The ENS regulates vital GI functions including gut motility, blood flow, fluid secretion, and absorption and thus maintains gut homeostasis. During vertebrate development it originates predominantly from the vagal neural crest (NC), a multipotent cell population that emerges from the caudal hindbrain region, migrates to and within the gut to ultimately generate neurons and glia in response to gut-derived signals. Loss of GI innervation due to congenital or acquired defects in ENS development causes enteric neuropathies which lack curative treatment. Human pluripotent stem cells (hPSCs) offer a promising in vitro source of enteric neurons for modeling human ENS development and pathology and potential use in cell therapy applications. Here we describe in detail a differentiation strategy for the derivation of enteric neural progenitors and neurons from hPSCs through a vagal NC intermediate. Using a combination of instructive signals and retinoic acid in a dose/time dependent manner, vagal NC cells commit into the ENS lineage and develop into enteric neurons and glia upon culture in neurotrophic media. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of vagal neural crest/early ENS progenitors from hPSCs Basic Protocol 2: Differentiation of hPSC-derived vagal NC/early ENS progenitors to enteric neurons and glia.

The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus it is a critical parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine and disease modelling applications. However, thein vitrogeneration of posterior spinal cord MNs has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), thebona fideprecursors of the mammalian spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well-defined. Here we determine the signals guiding the transition of human NMP-like cells toward posterior ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal early neural state while suppression of TGFβ-BMP signalling pathways, combined with SHH stimulation, promotes a ventral identity. Based on these results, we define an optimised protocol for the generation of posterior MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the functional comparison of hPSC-derived spinal cord cells of distinct axial identities.

The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the

AGF was supported by a Vice-Chancellor's Fellowship from the University of Sheffield, LJS was supported by a Chancellor's Fellowship from the University of Edinburgh. The deluge of single-cell data obtained by sequencing, imaging and epigenetic markers has led to an increasingly detailed description of cell state. However, it remains challenging to identify how cells transition between different states, in part because data are typically limited to snapshots in time. A prerequisite for inferring cell state transitions from such snapshots is to distinguish whether transitions are coupled to cell divisions. To address this, we present two minimal branching process models of cell division and differentiation in a well-mixed population. These models describe dynamics where differentiation and division are coupled or uncoupled. For each model, we derive analytic expressions for each subpopulation's mean and variance and for the likelihood, allowing exact Bayesian parameter inference and model selection in the idealised case of fully observed trajectories of differentiation and division events. In the case of snapshots, we present a sample path algorithm and use this to predict optimal temporal spacing of measurements for experimental design. We then apply this methodology to an in vitro dataset assaying the clonal growth of epiblast stem cells in culture conditions promoting self-renewal or differentiation. Here, the larger number of cell states necessitates approximate Bayesian computation. For both culture conditions, our inference supports the model where cell state transitions are coupled to division. For culture conditions promoting differentiation, our analysis indicates a possible shift in dynamics, with these processes becoming more coupled over time. Publisher PDF

SummaryThe enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1-7. Defects in the development and function of the ENS give rise to a range of disorders, termed enteric neuropathies and include conditions such as Hirschsprung’s disease. Little is known about the signalling that specifies early ENS progenitors. This has, thus far, limited progress in the generation of enteric neurons from human Pluripotent Stem Cells (hPSCs) that could provide a useful tool for disease modelling and regenerative medicine. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of both vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neuronsin vitroand followingin vivotransplantation, achieving long-term colonisation of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

The neural crest (NC) is a multipotent embryonic cell population generating distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and therefore current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

The neural crest (NC) is a multipotent embryonic cell population that generates various cell types in an axial position-dependent manner. Cranial NC cells give rise to mesoectodermal derivatives, melanocytes, neurons, and glia whereas the vagal NC generates the enteric nervous system and trunk NC cells produce sympathetic neurons and neuroendocrine cells. An attractive approach for studying human NC biology and modeling NC-associated developmental disorders (neurocristopathies) involves the in vitro production of NC cells from human pluripotent stem cells (hPSCs). However, most conventional differentiation protocols generate predominantly cranial NC cells but fail to induce trunk NC cells. Here we describe a detailed protocol for the efficient in vitro generation of trunk NC cells and their derivatives from hPSCs. This relies on the induction of an intermediate cell population that exhibits neural and mesodermal potential, resembling the embryonic neuromesodermal progenitors, which generate the postcranial body axis in vivo. © 2019 by John Wiley & Sons, Inc.

SummaryChimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs.

The transcription factors Nanog and Oct4 regulate pluripotency in the pre-implantation epiblast and in derivative embryonic stem cells. During post-implantation development, the precise timing and mechanism of the loss of pluripotency is unknown. Here, we show that in the mouse, pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis expression of both Nanog and Oct4 is regionalized. We show that pluripotency tracks the in vivo level of Oct4 and not Nanog by assessing the ability to reactivate or maintain Nanog expression in cell culture. Enforced Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitates moribund pluripotency. Our data suggest that decreasing Oct4 expression is converted to a sudden drop in competence to maintain pluripotency gene regulatory network activity that is subsequently stabilized by epigenetic locks.

Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.

Delivering the goods: By coupling proteins to varyingly sized polymeric microspheres, it is possible to deliver them to cells in an easy and effective way. For this study a fluorescent protein (EGFP) and a functional enzyme (β-galactosidase) were coupled to these particles. Evaluation of the cellular uptake after “beadfection” shows that the functionality and activity of these proteins were not adversely affected through coupling to the carrier system; this shows that their functional structure is retained.

Background Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency.

Timed pulses of WNT and FGF signaling convert human and mouse pluripotent stem cells into neuromesodermal progenitors that can be directed to differentiate into spinal cord and paraxial mesoderm cells, Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm in vitro and in vivo. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types in vitro and the analysis of previous difficult to access aspects of embryo development., Author Summary Stem cells are providing insight into embryo development and offering new approaches to clinical and therapeutic research. In part this progress arises from “directed differentiation” – artificially controlling the types of cells produced from stem cells. Here we describe the directed differentiation of mouse and human pluripotent stem cells into cells of the spinal cord and paraxial mesoderm (the tissue that generates muscle and bone that is normally found adjacent to the spinal cord). During embryo development, spinal cord and paraxial mesoderm arise from a shared group of precursors known as neuromesodermal progenitors (NMPs). We show that signals to which NMPs are exposed in embryos can be used to generate NMPs from pluripotent stem cells in a dish. We define conditions for the conversion of these NMPs into either spinal cord or mesoderm cells. Using these conditions, we provide evidence that the decision between spinal cord and mesoderm involves a gene, Brachyury, that promotes mesoderm production by inhibiting spinal cord generation. Together the data illustrate how mimicking normal embryonic development allows the generation of specific cell types from stem cells and that this can be used to analyse cells that are otherwise difficult to study.

Gene trapping is a technology originally developed for the simultaneous identification and mutation of genes by random integration in embryonic stem (ES) cells. While gene trapping was developed before efficient and high-throughput gene targeting, a significant proportion of the publically available mutant ES cell lines and mice were generated through a number of large-scale gene trapping initiatives. Moreover, elements of gene trap vectors continue to be incorporated into gene targeting vectors as a means to increase the efficiency of homologous recombination. Here, we review the current state of gene trapping technology and the applications of specific types of gene trap vector. As a component of this analysis, we consider the behavior of specific vector types both from the perspective of their application and how they can inform our annotation of the mammalian transcriptome. We consider the utility of gene trap vectors as tools for cell-based expression analysis, targeted screening in embryonic differentiation, and for use in cell lines derived from different lineages.

Promoterless gene trap vectors have been\ud widely used for high-efficiency gene targeting and\ud random mutagenesis in embryonic stem (ES) cells.\ud Unfortunately, such vectors are only effective\ud for genes expressed in ES cells and this has prompted\ud the development of expression-independent\ud vectors. These polyadenylation (poly A) trap vectors\ud employ a splice donor to capture an endogenous\ud gene’s polyadenylation sequence and provide\ud transcript stability. However, the spectrum of\ud mutations generated by these vectors appears\ud largely restricted to the last intron of target loci\ud due to nonsense-mediated mRNA decay (NMD)\ud making them unsuitable for gene targeting applications.\ud Here, we present novel poly A trap vectors\ud that overcome the effect of NMD and also employ\ud RNA instability sequences to improve splicing\ud efficiency. The set of random insertions generated\ud with these vectors show a significantly reduced\ud insertional bias and the vectors can be targeted\ud directly to a 5’ intron. We also show that this\ud relative positional independence is linked to the\ud human b-actin promoter and is most likely a result\ud of its transcriptional activity in ES cells. Taken\ud together our data indicate that these vectors are\ud an effective tool for insertional mutagenesis that\ud can be used for either gene trapping or gene\ud targeting.

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.