Fate Choice Research Articles

108 Defining a bi-stable network switch that governs stem cell self-renewal and differentiation in squamous cell carcinoma

Stem cell – like tumor propagating cells self-renew to drive clonal expansion or differentiate into post-mitotic cells without tumorigenic potential in squamous cell carcinomas. This fate choice is governed by a transcriptional network comprised of SOX2-PITX1-TP63 driven self-renewal and KLF4 dependent differentiation circuits. Yet, how stem cell – like tumor propagating cells switch from self-renewal to differentiation remains elusive. Here, we report that this cell fate choice is governed by a bi-stable Klf4 enhancer that is occupied by the transcription factor SOX2 in self-renewing or KLF4 in differentiating squamous cell carcinoma cells, dependent on whether SOX2 is phosphorylated.

Direct regulation of TCR rearrangement and expression by E proteins during early T cell development.

γδ T cells are widely distributed throughout mucosal and epithelial cell-rich tissues and are an important early source of IL-17 in response to several pathogens. Like αβ T cells, γδ T cells undergo a stepwise process of development in the thymus that requires recombination of genome-encoded segments to assemble mature T cell receptor (TCR) genes. This process is tightly controlled on multiple levels to enable TCR segment assembly while preventing the genomic instability inherent in the double-stranded DNA breaks that occur during this process. Each TCR locus has unique aspects in its structure and requirements, with different types of regulation before and after the αβ/γδ T cell fate choice. It has been known that Runx and Myb are critical transcriptional regulators of TCRγ and TCRδ expression, but the roles of E proteins in TCRγ and TCRδ regulation have been less well explored. Multiple lines of evidence show that E proteins are involved in TCR expression at many different levels, including the regulation of Rag recombinase gene expression and protein stability, induction of germline V segment expression, chromatin remodeling, and restriction of the fetal and adult γδTCR repertoires. Importantly, E proteins interact directly with the cis-regulatory elements of the TCRγ and TCRδ loci, controlling the predisposition of a cell to become an αβ T cell or a γδ T cell, even before the lineage-dictating TCR signaling events. This article is categorized under: Immune System Diseases > Stem Cells and Development Immune System Diseases > Genetics/Genomics/Epigenetics.

NR2F1 regulates a Schwann cell precursor-vs-melanocyte cellfate switch in a mouse model of Waardenburg syndrome type IV.

Waardenburg syndrome type 4 (WS4) combines abnormal development of neural crest cell (NCC)-derived melanocytes (causing depigmentation and inner ear dysfunction) and enteric nervous system (causing aganglionic megacolon). The full spectrum of WS4 phenotype is present in Spot mice, in which an insertional mutation close to a silencer element leads to NCC-specific upregulation of the transcription factor-coding gene Nr2f1. These mice were previously found to develop aganglionic megacolon because of NR2F1-induced premature differentiation of enteric neural progenitors into enteric glia. Intriguingly, this prior work also showed that inner ear dysfunction in Spot mutants specifically affects balance but not hearing, consistent with the absence of melanocytes in the vestibule only. Here, we report an analysis of the effect of Nr2f1 upregulation on the development of both inner ear and skin melanocytes, also taking in consideration their origin relative to the dorsolateral and ventral NCC migration pathways. In the trunk, we found that NR2F1 overabundance in Spot NCCs forces dorso-laterally migrating melanoblasts to abnormally adopt a Schwann cell precursor (SCP) fate and conversely prevents ventrally migrating SCPs to normally adopt a melanoblast fate. In the head, Nr2f1 upregulation appears not to be uniform, which might explain why SCP-derived melanocytes do colonize the cochlea while non-SCP-derived melanocytes cannot reach the vestibule. Collectively, these data point to a key role for NR2F1 in the control of SCP-vs-melanocyte fate choice and unveil a new pathogenic mechanism for WS4. Moreover, our data argue against the proposed existence of a transit-amplifying compartment of melanocyte precursors in hair follicles.

Macrophage-derived apoptotic vesicles regulate fate commitment of mesenchymal stem cells via miR155

BackgroundIn tissue engineering, mesenchymal stem cells (MSCs) are common seed cells because of abundant sources, strong proliferation ability and immunomodulatory function. Numerous researches have demonstrated that MSC-macrophage crosstalk played a key role in the tissue engineering. Macrophages could regulate the differentiation of MSCs via different molecular mechanisms, including extracellular vesicles. Apoptotic macrophages could generate large amounts of apoptotic vesicles (apoVs). ApoVs are rich in proteins, RNA (microRNAs, mRNAs, ncRNAs, etc.) and lipids, and are a key intercellular communication mediator that can exert different regulatory effects on recipient cells. MiRNAs account for about half of the total RNAs of extracellular vesicles, and play important roles in biological processes such as cell proliferation and differentiation, whereas the functions of macrophage-derived apoVs remain largely unknown. There was no research to clarify the role of macrophage-derived apoVs in MSC fate choices. In this study, we aimed to characterize macrophage-derived apoVs, and investigate the roles of macrophage-derived apoVs in the fate commitment of MSCs.MethodsWe characterized macrophage-derived apoVs, and investigated their role in MSC osteogenesis and adipogenesis in vitro and in vivo. Furthermore, we performed microRNA loss- and gain-of-function experiments and western blot to determine the molecular mechanism.ResultsMacrophages could produce a large number of apoVs after apoptosis. MSCs could uptake apoVs. Then, we found that macrophage-derived apoVs inhibited osteogenesis and promoted adipogenesis of MSCs in vitro and in vivo. In mechanism, apoVs were enriched for microRNA155 (miR155), and apoVs regulated osteogenesis and adipogenesis of MSCs by delivering miR155. Besides, miR155 regulated osteogenesis and adipogenesis of MSCs cultured with macrophage-derived apoVs via the SMAD2 signaling pathway.ConclusionsMacrophage-derived apoVs could regulate the osteogenesis and adipogenesis of MSCs through delivering miR155, which provided novel insights for MSC-mediated tissue engineering.

Identification of candidate genes simultaneously shared by adipogenesis and osteoblastogenesis from human mesenchymal stem cells.

In osteoporosis field, it had been clinically well established a given relationship between bone formation and lipid accumulation. Although numerous molecules had been well documented for adipogenesis and osteoblastogenesis (adipo-osteoblastogenesis), the reciprocal transcriptional regulation still remains to be explored. Here, we tried to identify the common candidate genes of adipocyte/osteoblastocyte differentiation at 3, 5, and 7 days using human mesenchymal stem cells (hMSCs) via RNA-Seq technique. By using RNA interference (RNAi), we further confirmed the function of candidate genes during adipo-osteoblastogenesis through Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). The identified 275 significantly differentially expressed genes (DEGs), especially with the down-regulated genes most prevalent and PI3K-AKT signaling pathway mostly enriched, were simultaneously shared by both differentiation events. Using lentiviral system, we further confirmed that ANKRD1 (ankyrin repeat domain 1) promoted adipogenesis and inhibited osteoblastogenesis via RNA interference (RNAi), and IGF1 (insulin like growth factor 1) simultaneously facilitated adipo-osteoblastogenesis on the base of gene expression of biomarkers and cellular phenotype property. This study would provide the potential molecular switches to control the adipocyte/osteoblastocyte balance or hMSCs fate choices and clues to screen the study and therapy targets of metabolic bone disease osteoporosis.

Ubiquitin-Specific Protease 22 Promotes Neural Stem Cells Stemness Maintenance and Adult Hippocampal Neurogenesis, Contributing to Cognitive Recovery Following Traumatic Brain Injury

Ubiquitin-specific protease 22 (USP22), a potential marker of cancer stem cells, significantly influences stem cell fate choices. However, its functions in neural stem cells (NSCs) and adult neurogenesis, especially following traumatic brain injury (TBI), remain only partially understood. Here, we found that aberrant USP22 expression could affect NSC proliferation and stemness maintenance, as assessed by the generation of neurospheres, cell counting kit-8 (CCK-8) and immunofluorescence staining in vitro. Moreover, USP22 depletion promotes the differentiation of NSCs, both in vitro and in vivo. In contrast, USP22 overexpression inhibits NSC differentiation into neurons. Interestingly, our data showed that USP22 promotes the proliferation but inhibits the differentiation of NSCs in the dentate gyrus (DG) of the hippocampus soon after TBI. The Morris water maze (MWM) test was adopted to evaluate neurological function, which confirmed that USP22 could improve the learning and memory capacity that was already compromised following TBI. Overall, this study uncovers a potentially novel regulatory role of USP22 in the proliferation and differentiation ability of NSCs, contributing to the hippocampus-dependent cognitive function of TBI mice and may be a novel target for future therapeutic approaches.

Insulin-like Growth Factor 2 Promotes Tissue-Specific Cell Growth, Proliferation and Survival during Development of Helicoverpa armigera.

During development, cells constantly undergo fate choices by differentiating, proliferating, and dying as part of tissue remodeling. However, we only begin to understand the mechanisms of these different fate choices. Here, we took the lepidopteran insect Helicoverpa armigera, the cotton bollworm, as a model to reveal that insulin-like growth factor 2 (IGF-2-like) prevented cell death by promoting cell growth and proliferation. Tissue remodeling occurs during insect metamorphosis from larva to adult under regulation by 20-hydroxyecdysone (20E), a steroid hormone. An unknown insulin-like peptide in the genome of H. armigera was identified as IGF-2-like by sequence analysis using human IGFs. The expression of Igf-2-like was upregulated by 20E. IGF-2-like was localized in the imaginal midgut during tissue remodeling, but not in larval midgut that located nearby. IGF-2-like spread through the fat body during fat body remodeling. Cell proliferation was detected in the imaginal midgut and some fat body cells expressing IGF-2-like. Apoptosis was detected in the larval midgut and some fat body cells that did not express IGF-2-like, suggesting the IGF-2-like was required for cell survival, and IGF-2-like and apoptosis were exclusive, pointing to a survival requirement. Knockdown of Igf-2-like resulted in repression of growth and proliferation of the imaginal midgut and fat body. Our results suggested that IGF-2-like promotes cell growth and proliferation in imaginal tissues, promoting cell death avoidance and survival of imaginal cells during tissue remodeling. It will be interesting to determine whether the mechanism of action of steroid hormones on insulin growth factors is conserved in other species.

A Nuclear Belt Fastens on Neural Cell Fate.

Successful embryonic and adult neurogenesis require proliferating neural stem and progenitor cells that are intrinsically and extrinsically guided into a neuronal fate. In turn, migration of new-born neurons underlies the complex cytoarchitecture of the brain. Proliferation and migration are therefore essential for brain development, homeostasis and function in adulthood. Among several tightly regulated processes involved in brain formation and function, recent evidence points to the nuclear envelope (NE) and NE-associated components as critical new contributors. Classically, the NE was thought to merely represent a barrier mediating selective exchange between the cytoplasm and nucleoplasm. However, research over the past two decades has highlighted more sophisticated and diverse roles for NE components in progenitor fate choice and migration of their progeny by tuning gene expression via interactions with chromatin, transcription factors and epigenetic factors. Defects in NE components lead to neurodevelopmental impairments, whereas age-related changes in NE components are proposed to influence neurodegenerative diseases. Thus, understanding the roles of NE components in brain development, maintenance and aging is likely to reveal new pathophysiological mechanisms for intervention. Here, we review recent findings for the previously underrepresented contribution of the NE in neuronal commitment and migration, and envision future avenues for investigation.

An enhancer located in a Pde6c intron drives transient expression in the cone photoreceptors of developing mouse and human retinas

How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans.

Cell-Cell Contact Mediates Gene Expression and Fate Choice of Human Neural Stem/Progenitor Cells.

Transplantation of Neural Stem/Progenitor Cells (NPCs) is a promising regenerative strategy to promote neural repair following injury and degeneration because of the ability of these cells to proliferate, migrate, and integrate with the host tissue. Precise in vitro control of NPC proliferation without compromising multipotency and differentiation ability is critical in stem cell maintenance. This idea was highlighted in recent clinical trials, where discrepancies in NPC culturing protocols produced inconsistent therapeutic benefits. Of note, cell density plays an important role in regulating the survival, proliferation, differentiation, and fate choice of stem cells. To determine the extent of variability produced by inconsistent culturing densities, the present study cultured human-induced pluripotent NPCs (hiPSC-NPCs) at either a low or high plating density. hiPSC-NPCs were then isolated for transcriptomic analysis or differentiation in vitro. Following sequencing analysis, genes involved in cell–cell contact-mediated pathways, including Hippo-signaling, NOTCH, and WNT were differentially expressed. Modulation of these pathways was highly associated with the regulation of pro-neuronal transcription factors, which were also upregulated in response to higher-density hiPSC-NPC culture. Moreover, higher plating density translated into a greater neuronal and less astrocytic differentiation in vitro. This study highlights the importance of precisely controlling culture conditions during the development of NPC transplantation therapies.

Epigenetic Control of Muscle Stem Cells: Focus on Histone Lysine Demethylases.

Adult skeletal muscle is mainly composed of post-mitotic, multinucleated muscle fibers. Upon injury, it has the unique ability to regenerate thanks to the activation of a subset of quiescent muscle stem cells (MuSCs). Activated MuSCs either differentiate to repair muscle, or self-renew to maintain the pool of MuSC. MuSC fate determination is regulated by an intricate network of intrinsic and extrinsic factors that control the expression of specific subsets of genes. Among these, the myogenic regulatory factors (MRFs) are key for muscle development, cell identity and regeneration. More globally, cell fate determination involves important changes in the epigenetic landscape of the genome. Such epigenetic changes, which include DNA methylation and post-translational modifications of histone proteins, are able to alter chromatin organization by controlling the accessibility of specific gene loci for the transcriptional machinery. Among the numerous epigenetic modifications of chromatin, extensive studies have pointed out the key role of histone methylation in cell fate control. Particularly, since the discovery of the first histone demethylase in 2004, the role of histone demethylation in the regulation of skeletal muscle differentiation and muscle stem cell fate has emerged to be essential. In this review, we highlight the current knowledge regarding the role of histone demethylases in the regulation of muscle stem cell fate choice.

The ubiquitin ligase Cul5 regulates CD4+ T cell fate choice and allergic inflammation

Antigen encounter directs CD4+ T cells to differentiate into T helper or regulatory cells. This process focuses the immune response on the invading pathogen and limits tissue damage. Mechanisms that govern T helper cell versus T regulatory cell fate remain poorly understood. Here, we show that the E3 ubiquitin ligase Cul5 determines fate selection in CD4+ T cells by regulating IL-4 receptor signaling. Mice lacking Cul5 in T cells develop Th2 and Th9 inflammation and show pathophysiological features of atopic asthma. Following T cell activation, Cul5 forms a complex with CIS and pJak1. Cul5 deletion reduces ubiquitination and subsequent degradation of pJak1, leading to an increase in pJak1 and pSTAT6 levels and reducing the threshold of IL-4 receptor signaling. As a consequence, Cul5 deficient CD4+ T cells deviate from Treg to Th9 differentiation in low IL-4 conditions. These data support the notion that Cul5 promotes a tolerogenic T cell fate choice and reduces susceptibility to allergic asthma.

The E-Id Axis Instructs Adaptive Versus Innate Lineage Cell Fate Choice and Instructs Regulatory T Cell Differentiation.

Immune responses are primarily mediated by adaptive and innate immune cells. Adaptive immune cells, such as T and B cells, evoke antigen-specific responses through the recognition of specific antigens. This antigen-specific recognition relies on the V(D)J recombination of immunoglobulin (Ig) and T cell receptor (TCR) genes mediated by recombination-activating gene (Rag)1 and Rag2 (Rag1/2). In addition, T and B cells employ cell type-specific developmental pathways during their activation processes, and the regulation of these processes is strictly regulated by the transcription factor network. Among these factors, members of the basic helix-loop-helix (bHLH) transcription factor mammalian E protein family, including E12, E47, E2-2, and HEB, orchestrate multiple adaptive immune cell development, while their antagonists, Id proteins (Id1-4), function as negative regulators. It is well established that a majority of T and B cell developmental trajectories are regulated by the transcriptional balance between E and Id proteins (the E-Id axis). E2A is critically required not only for B cell but also for T cell lineage commitment, whereas Id2 and Id3 enforce the maintenance of naïve T cells and naïve regulatory T (Treg) cells. Here, we review the current knowledge of E- and Id-protein function in T cell lineage commitment and Treg cell differentiation.

Black ginseng ameliorates cellular senescence via p53/p21 pathway

BackgroundCellular senescence, one of the hallmarks of aging, refers to a permanent cell cycle arrest and is accelerated during the aging process. A senolytic, a small molecule that eliminates senescent cells, can be a potential strategy to extend healthy lifespan and ameliorate age‐associated diseases. Black ginseng (BG) prepared via nine‐times repetitive steaming and drying process from fresh ginseng (Panax ginsengC.A. Meyer) has been reported to its physiological benefits against reactive oxygen species (ROS), inflammation, and oncogenesis which are common cues to induce aging. Therefore, the present study was aimed to investigate the effect of BG on cellular senescence.MethodsSenescence‐associated β‐galactosidase (SA‐β‐gal) staining was performed on primary mouse embryonic fibroblasts (MEFs) treated 5 μg/mL BG extract for 7 days, and then senescence was induced by γ‐ray irradiation (20 Gy). For animal studies, C57BL/6 male mice were divided into three groups: 9‐week‐old mice orally administered distilled water (Young), 21‐month‐old mice received administration of distilled water (Old), and aged mice supplemented 300 mg/kg BG extracts (Old + BG). After 4 weeks of daily injection, PCR array and immunoblotting were conducted to evaluate mRNA expression and protein levels of targets associated with cellular senescence and cell cycle regulation in metabolic organs, liver, skeletal muscle (SKM), and white adipose tissue (WAT).ResultsIn in vitro study, BG treatment reduced the density of SA‐β‐gal‐positive MEFs after γ‐ray irradiation (Fig. 1). In aged mice study, BG supplementation remarkedly downregulated age‐related hepatic genes, especially in complement component 1q B (C1qb) and C (C1qc), compared to the aged control group. We further examined the canonical Wnt signaling, augmented in a mouse model of accelerated aging, since the complement C1q family activates Wnt signaling by binding to Frizzled receptors. By assessing the related markers, BG supplementation significantly decreased the hepatic β‐catenin expression level; it also slightly reduced the levels of glycogen synthase kinase 3β (GSK3β) and its phosphorylated form (p‐GSK3β). Recent studies have revealed that Wnt signaling involves in mammalian aging by regulating cell fate and proliferation choice. Thus, we evaluated the expressions of p53 and p21, which involves in cell cycle regulation, and both levels decreased in liver samples from Old + BG group compared to the Old group (Fig. 2). In support of this notion, BG treatment decreased the hepatic mRNA expression levels of senescence‐associated secretory phenotypes (SASPs) such as matrix metallopeptidase 12 (Mmp12) and chemokine ligand 2 (Cxcl2). Consistent with the results in the liver, other metabolic organs, SKM and WAT, showed significantly lower p53 expression in Old + BG group over the Old group.ConclusionThe current study indicates that BG could be a potential candidate of senolytics to reversely regulate cellular senescence, resulting from downregulated complement system affecting Wnt signaling pathways and p53/p21 pathway in metabolic organs.

Uncovering the Ciliogenesis Regulatory Network

Ciliopathies comprise disorders associated with aberrant function of essential organelles called cilia. Cilia are found in almost all vertebrate cells, with a subset of this population being motile and/or multiciliated. Over 1,000 proteins have been associated with the ciliary proteome, yet the regulatory circuit controlling the formation of cilia remains lesser known. Our lab has identified transcription factors and coactivators that contribute to the ciliogenesis circuit. Such factors include orphan nuclear receptor Esrrγ and coactivator Ppargc1a/PGC1a. Independently, these factors regulate both mono‐ and multi‐ciliated cells in the early signaling node, ear, and embryonic kidney (pronephros). Absence of these factors results in decreased multiciliated cells, decreased cilia length, and increased unciliated basal bodies. Considering these phenotypic similarities, we hypothesized a cooperative relationship between Esrrγ and Pppargc1a in the context of ciliated cell development. By modelling dual heterozygosity with genetic interaction studies, we found that ppargc1aworks synergistically with esrrγato regulate prostaglandin signaling to support ciliary outgrowth and multiciliated cell fate choice. While previous studies have alluded to the interaction of Esrrγ and Ppargc1a/PGC1a in metabolism, this is the first report of a cooperative effect that is essential for ciliogenesis or lineage decisions in development. This study has far‐reaching implications, as cilia are present in various tissues, and these factors may serve as therapeutic targets for ciliopathic conditions and birth defects.

A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices.

Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts.

Odontogenic MSC Heterogeneity: Challenges and Opportunities for Regenerative Medicine.

Cellular heterogeneity refers to the genetic and phenotypic differences among cells, which reflect their various fate choices, including viability, proliferation, self-renewal probability, and differentiation into different lineages. In recent years, research on the heterogeneity of mesenchymal stem cells has made some progress. Odontogenic mesenchymal stem cells share the characteristics of mesenchymal stem cells, namely, good accessibility, low immunogenicity and high stemness. In addition, they also exhibit the characteristics of vasculogenesis and neurogenesis, making them attractive for tissue engineering and regenerative medicine. However, the usage of mesenchymal stem cell subgroups differs in different diseases. Furthermore, because of the heterogeneity of odontogenic mesenchymal stem cells, their application in tissue regeneration and disease management is restricted. Findings related to the heterogeneity of odontogenic mesenchymal stem cells urgently need to be summarized, thus, we reviewed studies on odontogenic mesenchymal stem cells and their specific subpopulations, in order to provide indications for further research on the stem cell regenerative therapy.

The TH1 cell lineage-determining transcription factor T-bet suppresses TH2 gene expression by redistributing GATA3 away from TH2 genes.

Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3′s sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.

SALSA, a genetically encoded biosensor for spatiotemporal quantification of Notch signal transduction invivo.

Notch-mediated lateral specification is a fundamental mechanism to resolve stochastic cell fate choices by amplifying initial differences between equivalent cells. To study how stochastic events impact Notch activity, we developed a biosensor, SALSA (sensor able to detect lateral signaling activity), consisting of an amplifying "switch"-Notch tagged with TEV protease-and a "reporter"-GFP fused to a nuclearly localized red fluorescent protein, separated by a TEVp cut site. When ligand activates Notch, TEVp enters the nucleus and releases GFP from its nuclear tether, allowing Notch activation to be quantified based on the changes in GFP subcellular localization. We show that SALSA accurately reports Notch activity in different signaling paradigms in Caenorhabditis elegans and use time-lapse imaging to test hypotheses about how stochastic elements ensure a reproducible and robust outcome in a canonical lin-12/Notch-mediated lateral signaling paradigm. SALSA should be generalizable to other experimental systems and be adaptable to increase options for bespoke "SynNotch" applications.

Gene Regulatory Networks of Epidermal and Neural Fate Choice in a Chordate.

Neurons are a highly specialized cell type only found in metazoans. They can be scattered throughout the body or grouped together, forming ganglia or nerve cords. During embryogenesis, centralized nervous systems develop from the ectoderm, which also forms the epidermis. How pluripotent ectodermal cells are directed toward neural or epidermal fates, and to which extent this process is shared among different animal lineages, are still open questions. Here, by using micromere explants, we were able to define in silico the putative gene regulatory networks (GRNs) underlying the first steps of the epidermis and the central nervous system formation in the cephalochordate amphioxus. We propose that although the signal triggering neural induction in amphioxus (i.e., Nodal) is different from vertebrates, the main transcription factors implicated in this process are conserved. Moreover, our data reveal that transcription factors of the neural program seem to not only activate neural genes but also to potentially have direct inputs into the epidermal GRN, suggesting that the Nodal signal might also contribute to neural fate commitment by repressing the epidermal program. Our functional data on whole embryos support this result and highlight the complex interactions among the transcription factors activated by the signaling pathways that drive ectodermal cell fate choice in chordates.