Cell Fate Restriction Research Articles

Single-cell epigenomic reconstruction of developmental trajectories from pluripotency in human neural organoid systems

Cell fate progression of pluripotent progenitors is strictly regulated, resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study, we use human brain and retina organoid models and present single-cell profiling of H3K27ac, H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction, resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination.

The single-cell chromatin landscape in gonadal cell lineage specification

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency.

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.

Proteomic/transcriptomic analysis of erythropoiesis.

Erythropoiesis is a hierarchical process by which hematopoietic stem cells give rise to red blood cells through gradual cell fate restriction and maturation. Deciphering this process requires the establishment of dynamic gene regulatory networks (GRNs) that predict the response of hematopoietic cells to signals from the environment. Although GRNs have historically been derived from transcriptomic data, recent proteomic studies have revealed a major role for posttranscriptional mechanisms in regulating gene expression during erythropoiesis. These new findings highlight the need to integrate proteomic data into GRNs for a refined understanding of erythropoiesis. Here, we review recent proteomic studies that have furthered our understanding of erythropoiesis with a focus on quantitative mass spectrometry approaches to measure the abundance of transcription factors and cofactors during differentiation. Furthermore, we highlight challenges that remain in integrating transcriptomic, proteomic, and other omics data into a predictive model of erythropoiesis, and discuss the future prospect of single-cell proteomics. Recent proteomic studies have considerably expanded our knowledge of erythropoiesis beyond the traditional transcriptomic-centric perspective. These findings have both opened up new avenues of research to increase our understanding of erythroid differentiation, while at the same time presenting new challenges in integrating multiple layers of information into a comprehensive gene regulatory model.

Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis.

Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium.

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified protein phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hippo kinase, and consequently its substrate Warts, to release the transcriptional co-activator Yorkie into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.

Deconvolution of Hematopoietic Commitment Decisions By Genome-Wide Analysis of Progressive DNA Methylation Changes

Recent advances in single cell transcriptome analyses have resulted in the derivation of new models to describe the hierarchical organization of the mammalian hematopoietic system. While such an approach appears to be effective at discerning the trajectory of differentiation from hematopoietic stem cells (HSCs) to a given mature lineage, it remains a challenge to identify definitive points where specific lineage fates become restricted. The characterization of molecular events that correspond to such commitment decisions is critical to our interrogation of the existence and nature of serial bifurcation steps that are hypothesized to underlie the process of hematopoiesis. 5-Methylcytosine is a stable epigenetic modification, whose remodeling at specific CpG residues appears to be integral to the process of enforcing lineage-restricted gene expression programs. We have previously observed that the remodeling of the DNA methylome appears to be both progressive and irreversible during the process of hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. In order to pursue this concept further, we used tagmentation-based whole-genome bisulfite sequencing to generate a genome-wide DNA methylation map of murine hematopoiesis. This map encompasses 26 different FACS-purified populations, ranging from LT-HSCs through to terminally differentiated blood cell lineages. Across all cell populations studied, we identified 147,232 differentially methylated regions (DMRs). In line with our previous data, hierarchical clustering of these DMRs revealed coordinately regulated regions that show progressive and unidirectional lineage-specific DNA methylation dynamics during hematopoietic differentiation that would be indicative of a molecular mechanism of cell fate restriction. Single cell DNA methylome analysis indicated that methylation programming may be exclusive for a specific lineage within each cell analyzed, supporting the use of this data to establish the discreet points at which lineage commitment occurs. Along these lines, lineage-specific DMRs could already be identified within the early hematopoietic stem and multipotent progenitor compartments, supporting the concept that lineage restriction occurs early during differentiation and providing a potential molecular basis for so called lineage-priming/bias. Indeed, a phylogenetic tree of the entire hematopoietic system could be constructed purely based on methylation remodeling events that took place in the Lin-, Sca-1+, c-Kit+ compartment. To gain further insight into how the DNA methylation programming relates to regulation of gene expression, we generated a comprehensive single cell transcriptome map encompassing the entire hematopoietic component of the bone marrow. Integration of DNA methylation dynamics with single cell gene expression dynamics provided evidence for anti-correlation between the transcriptional program and DNA methylation. However, loss of DNA methylation was not invariably associated with an increase in gene expression, suggesting that DNA methylation has more a permissive rather than an instructive role in regulating gene expression programs. We next applied our data set to the exploration of the phenomenon of myeloid lineage bias that has been described in aged mice, by investigating the methylomes of young and aged HSCs. Compared to young HSCs, we identified 3,275 DMRs in aged HSCs, which were predominantly associated with loss of DNA methylation and affected genes involved in HSC adhesion and migration, such as Vwf and ITGB3. Remarkably, 46 % of aging DMRs overlapped with the methylome programs identified in hematopoietic differentiation and were enriched for genes involved in integrin signaling, platelet activation and aggregation. This data suggests that HSC aging results in remodeling of the DNA methylome in vivo, in a manner that is associated with a differentiation bias towards the megakaryocytic lineage. Together, our work provides a rich resource to investigate DNA methylation changes in normal or diseased hematopoiesis, across a broad range of conditions such as aging. Disclosures Lipka: InfectoPharm GmbH: Employment.

MLL1 Inhibition and Vitamin D Signaling Cooperate to Facilitate the Expanded Pluripotency State

SUMMARYDynamic establishment of histone modifications in early development coincides with programed cell fate restriction and loss of totipotency beyond the early blastocyst stage. Causal function of histone-modifying enzymes in this process remains to be defined. Here we show that inhibiting histone methyl-transferase MLL1 reprograms naive embryonic stem cells (ESCs) to expanded pluripotent stem cells (EPSCs), with differentiation potential toward both embryonic and extraembryonic lineages in vitro and in vivo. MLL1 inhibition or deletion upregulates gene signatures of early blastomere development. The function of MLL1 in restricting induction of EPSCs is mediated partly by Gc, which regulates cellular response to vitamin D signaling. Combined treatment of MLL1 inhibitor and 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3) cooperatively enhanced functionality of EPSCs, triggering an extended 2C-like state in vitro and robust totipotent-like property in vivo. Our study sheds light on interplay between epigenetics and vitamin D pathway in cell fate determination.

1019 - DECONVOLUTION OF HEMATOPOIETIC COMMITMENT DECISIONS BY GENOME-WIDE ANALYSIS OF PROGRESSIVE DNA METHYLATION CHANGES

Advances in single cell transcriptome analyses have resulted in the derivation of new models to describe the hierarchical organization of the mammalian hematopoietic system. While such an approach appears to be effective at discerning the trajectory of differentiation from hematopoietic stem cells (HSCs) to a given mature lineage, it remains a challenge to identify definitive points where specific lineage fates become restricted. 5-MeC is a stable epigenetic modification, whose remodeling is integral to the process of enforcing lineage-restricted gene expression programs. We have previously observed that the remodeling of the DNA methylome appears to be both progressive and irreversible during the process of hematopoietic differentiation, suggesting that this modification could be used to unambiguously identify molecular marks of lineage commitment. To pursue this concept, we generated a genome-wide DNA methylation map of murine hematopoiesis. This map, comprised of a total of 147,232 differentially methylated regions (DMRs), encompasses 26 different FACS-purified populations. Analysis of these DMRs revealed progressive lineage-specific DNA methylation dynamics that would be indicative of a molecular mechanism of cell fate restriction. Lineage-specific DMRs could already be identified within the early hematopoietic stem and multipotent progenitor compartments, supporting the concept that lineage restriction occurs early during differentiation. In contrast with previous studies, integration of our methylome data with single cell gene expression data encompassing the entire hematopoietic component of the bone marrow, revealed an unprecedented degree of correlation between the transcriptional program and DNA methylation levels. Both inflammatory challenge and ageing resulted in remodeling of the HSC DNA methylome in vivo, in a manner that was not predominantly associated with priming towards any particular differentiated cell fate. Rather the reprogrammed methylome may represent an epigenetic memory of gene expression programs resulting from extrinsic stimuli.

Maps of variability in cell lineage trees.

New approaches to lineage tracking have allowed the study of differentiation in multicellular organisms over many generations of cells. Understanding the phenotypic variability observed in these lineage trees requires new statistical methods. Whereas an invariant cell lineage, such as that for the nematode Caenorhabditis elegans, can be described by a lineage map, defined as the pattern of phenotypes overlaid onto the binary tree, a traditional lineage map is static and does not describe the variability inherent in the cell lineages of higher organisms. Here, we introduce lineage variability maps which describe the pattern of second-order variation in lineage trees. These maps can be undirected graphs of the partial correlations between every lineal position, or directed graphs showing the dynamics of bifurcated patterns in each subtree. We show how to infer these graphical models for lineages of any depth from sample sizes of only a few pedigrees. This required developing the generalized spectral analysis for a binary tree, the natural framework for describing tree-structured variation. When tested on pedigrees from C. elegans expressing a marker for pharyngeal differentiation potential, the variability maps recover essential features of the known lineage map. When applied to highly-variable pedigrees monitoring cell size in T lymphocytes, the maps show that most of the phenotype is set by the founder naive T cell. Lineage variability maps thus elevate the concept of the lineage map to the population level, addressing questions about the potency and dynamics of cell lineages and providing a way to quantify the progressive restriction of cell fate with increasing depth in the tree.

Ubiquitination-Independent Repression of PRC1 Targets during Neuronal Fate Restriction in the Developing Mouse Neocortex

Polycomb repressive complex (PRC) 1 maintains developmental genes in a poised state through monoubiquitination (Ub) of histone H2A. Although Ub-independent functions of PRC1 have also beensuggested, it has remained unclear whether Ub-dependent and -independent functions of PRC1 operate differentially in a developmental context. Here, we show that the E3 ubiquitin ligase activity of Ring1B, a core component of PRC1, is necessary for the temporary repression of key neuronal genes in neurogenic (early-stage) neural stem or progenitor cells (NPCs) but is dispensable for the persistent repression of these genes associated with the loss of neurogenic potential in astrogliogenic (late-stage) NPCs. Our results also suggest that histone deacetylase (HDAC) activity of the NuRD/MBD3 complex and Phc2-dependent PRC1 clustering are necessary for the transition from the Ub-dependent to -independent function of PRC1. Together, these results indicate that Ub-independent mode of repression by PRC1 plays a key role in mammalian development during cell fate restriction.

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

The cis-regulatory dynamics of embryonic development at single-cell resolution.

Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring single cell gene expression are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain elusive. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single cell resolution. Using single cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq)1, we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila embryos spanning three landmark embryonic stages: 2-4 hours (hrs) after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises ~6,000 multipotent cells; 6-8hrs (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12hrs (predominantly stage 13), when each of the embryo’s >20,000 cells are undergoing terminal differentiation. Our results reveal spatial heterogeneity in the usage of the regulatory genome prior to gastrulation, a feature that aligns with future cell fate, and nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility, while maintaining a signature of their germ layer of origin. The data reveal overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program reminiscent of the mesendoderm lineage in other species2–4. Altogether, we identify over 30,000 distal regulatory elements exhibiting tissue-specific accessibility. We validated the germ layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving 90% accuracy. Overall, our results demonstrate the power of shotgun single cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.

Defined Culture Conditions Accelerate Small-molecule-assisted Neural Induction for the Production of Neural Progenitors from Human-induced Pluripotent Stem Cells

The use of defined conditions for derivation, maintenance, and differentiation of human-induced pluripotent stem cells (hiPSCs) provides a superior experimental platform to discover culture responses to differentiation cues and elucidate the basic requirements for cell differentiation and fate restriction. Adoption of defined systems for reprogramming, undifferentiated growth, and differentiation of hiPSCs was found to significantly influence early stage differentiation signaling requirements and temporal kinetics for the production of primitive neuroectoderm. The bone morphogenic protein receptor agonist LDN-193189 was found to be necessary and sufficient for neural induction in a monolayer system with landmark antigens paired box 6 and sex-determining region Y-box 1 appearing within 72 h. Preliminary evidence suggests this neuroepithelium was further differentiated to generate ventral spinal neural progenitors that produced electrophysiologically active neurons in vitro, maintaining viability posttransplantation in an immunocompromised host. Our findings support current developments in the field, demonstrating that adoption of defined reagents for the culture and manipulation of pluripotent stem cells is advantages in terms of simplification and acceleration of differentiation protocols, which will be critical for future clinical translation.

Cellular plasticity in cardiovascular development and disease.

Knowledge on cellular differentiation pathways is critical to understanding organ development, homeostasis, and disease. Studying cell differentiation and cell fate restrictions in these contexts can be done through lineage tracing experiments, which entail permanent labeling of a cell and all its progeny. Recent lineage experiments within the cardiovascular system have uncovered unexpected findings on cellular origins during organogenesis and cell plasticity during disease. For example, there is increasing evidence that multiple progenitor sources exist for a single cell type, and that cells have remarkable expansive capacities under disease settings. Here, we summarize some recent findings in the cardiovascular system and highlight where there is evidence that the underlying concepts are a widespread phenomenon used by other organ systems. Developmental Dynamics 246:328-335, 2017. © 2016 Wiley Periodicals, Inc.

Functional constraints on SoxE proteins in neural crest development: The importance of differential expression for evolution of protein activity

Vertebrate SoxE genes (Sox8, 9, and 10) are key regulators of neural crest cell (NCC) development. These genes arose by duplication from a single SoxE gene in the vertebrate ancestor. Although SoxE paralogs are coexpressed early in NCC development, later, Sox9 is restricted to skeletogenic lineages in the head, and Sox10 to non-skeletogenic NCC in the trunk and head. When this subfunctionalization evolved and its possible role in the evolution of the neural crest are unknown. Sea lampreys are basal vertebrates that also possess three SoxE genes, while only a single SoxE is present in the cephalochordate amphioxus. In order to address the functional divergence of SoxE genes, and to determine if differences in their biochemical functions may be linked to changes in neural crest developmental potential, we examined the ability of lamprey and amphioxus SoxE genes to regulate differentiation of NCC derivatives in zebrafish colourless (cls) mutants lacking expression of sox10. Our findings suggest that the proto-vertebrate SoxE gene possessed both melanogenic and neurogenic capabilities prior to SoxE gene duplication. Following the agnathan-gnathostome split, lamprey SoxE1 and SoxE3 largely lost their melanogenic and/or enteric neurogenic properties, while gnathostome SoxE paralogs have retained functional conservation. We posit that this difference in protein subfunctionalization is a direct consequence of the independent regulation of SoxE paralog expression between the two lineages. Specifically, we propose that the overlapping expression of gnathostome SoxE paralogs in early neural crest largely constrained the function of gnathostome SoxE proteins. In contrast, the largely non-overlapping expression of lamprey SoxE paralogs allowed them to specialize with regard to their DNA-binding and/or protein interaction properties. Restriction of developmental potential among cranial and trunk neural crest in lampreys may be related to constraints on SoxE activity among duplicates, but such specialization does not appear to have occurred in gnathostomes. This highlights an important difference in the evolution of SoxE activity between these two divergent vertebrate lineages and provides insights for understanding how cell fate restriction in different NCC populations may be dependent on subfunctionalization among SoxE duplicates.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

BackgroundA central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties.ResultsWe demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation.ConclusionsWe conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0267-0) contains supplementary material, which is available to authorized users.

Regulation of Differentiation by Calcium-Sensing Receptor in Normal and Tumoral Developing Nervous System

During normal development of the nervous system (NS), neural progenitor cells (NPCs) produce specialized populations of neurons and glial cells upon cell fate restriction and terminal differentiation. These sequential processes require the dynamic regulation of thousands of genes. The calcium-sensing receptor (CaSR) is temporally and spatially regulated in both neurons and glial cells during development of the NS. In particular, CaSR expression and function have been shown to play a significant role during differentiation of NPCs toward the oligodendrocyte lineage and also in maturation of cerebellar granule cell precursors (GCPs). Moreover, CaSR regulates axonal and dendritic growth in both central and peripheral nervous systems (PNSs), a process necessary for proper construction of mature neuronal networks. On the other hand, several lines of evidence support a role for CaSR in promotion of cell differentiation and inhibition of proliferation in neuroblastoma, a tumor arising from precursor cells of developing PNS. Thus, among the variety of NS functions in which the CaSR participates, this mini-review focuses on its role in differentiation of normal and tumoral cells. Current knowledge of the mechanisms responsible for CaSR regulation and function in these contexts is also discussed, together with the therapeutic opportunities provided by CaSR allosteric modulators.

Overcoming CD4 T helper cell fate restrictions to enhance CD8 T cell and B cell immunity to control persistent LCMV infection

Abstract Persistent viral infection inhibits CD4 Th1 responses, resulting in loss of the CD8 T cell helping Th1 subset and accumulation of B cell helping Tfh cells. Yet, the mechanisms that inhibit CD4 Th1 differentiation and the repercussions of loss of Th1 cells on viral control are unclear. Herein, we demonstrate that the dual upregulation of PD-L1 and IL-10 by chronic type I interferon (IFN-I) signaling during persistent infection suppresses CD4 Th1 priming. Therapeutic blockade of IL-10R and PD-L1 could restore the differentiation of new CD4 Th1 cells, highlighting a new function for PD-L1 to co-inhibit CD4 Th1 priming and identifying a new combinatorial suppressive mechanism during persistent infection. However, CD4 Th1 cells generated by blocking IFN-I signaling in vivo could not be sustained, giving way to a Tfh based response and predominant help to B cells. In vitro polarized virus-specific CD4 Th1 cells also converted towards Tfh cells, however maintained a population of Th1 cells that could enhance exhausted CD8 T cell responses and promote augmented viral clearance. Interestingly, virus-specific CD4 Th17 and Treg cells were also redirected towards Tfh as persistent infection progressed. Thus, our studies have important implications for stability of de novo activated and immunotherapeutic T cells and demonstrate that the redirection away from Th1 responses is a mechanism of immunosuppression to limit the control of persistent viral infection.

Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish.

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.