Single-cell lineage mapping of a diverse virus-specific naive CD4 T cell repertoire

Tracking how individual naive T cells from a natural TCR repertoire clonally expand, differentiate, and make lineage choices in response to an infection has not previously been possible. Here, using single-cell sequencing technology to identify clones by their unique TCR sequences, we were able to trace the clonal expansion, differentiation trajectory, and lineage commitment of individual virus-specific CD4 T cells during an acute lymphocytic choriomeningitis virus (LCMV) infection. Notably, we found previously unappreciated clonal diversity and cellular heterogeneity among virus-specific helper T cells. Interestingly, although most naive CD4 T cells gave rise to multiple lineages at the clonal level, ∼28% of naive cells exhibited a preferred lineage choice toward either Th1 or TFH cells. Mechanistically, we found that TCR structure, in particular the CDR3 motif of the TCR α chain, skewed lineage decisions toward the TFH cell fate.

Rapid and efficient in vivo astrocyte-to-neuron conversion with regional identity and connectivity?

ABSTRACTIn vivo reprogramming of glia into functional neurons emerges as potential regeneration-based therapeutics for neural injuries or neurological diseases. Recent studies show that AAV-based manipulation of certain factors can rapidly and highly efficiently convert resident glia into functional neurons with brain region-specificity and precise connectivity. Using NEUROD1 as an example, we here show that the presumed astrocytes-converted neurons are essentially endogenous neurons in the adult mouse brain. AAV-mediated co-expression of NEUROD1 and a reporter indeed specifically, rapidly, and efficiently induces numerous reporter-labeled neurons. However, these neurons cannot be traced back to quiescent or reactive astrocytes by using stringent lineage-mapping strategies. Conversely, reporter-labeled neurons cannot be detected when NEUROD1 is strictly expressed in adult brain astrocytes. Through a retrograde labeling approach, our results rather reveal that endogenous neurons are the cell source for NEUROD1-induced reporter-labeled neurons. These results underline the indispensable value of stringent lineage-tracing strategies and beg for cautious interpretation of the in vivo reprogramming phenomena.

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed >95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P<0.01, n>4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

Conservation and Divergence of Related Neuronal Lineages in the Drosophila Central Brain

SummaryWiring a complex brain requires enormous cell specificity. This specificity is laid out via a developmental process where neural stem cells produce countless diverse neurons. To help elucidate this process and resolve the considerable dynamic specificity, we need to observe the development of multiple neuronal lineages. Drosophila central brain lineages are predetermined, comprised of a fixed set of neurons born in pairs in a specific order. To reveal specific roles of lineage identity, Notch-dependent sister fate specification, and temporal patterning in morphological diversification, we mapped approximately one quarter of the Drosophila central brain lineages. While we found large aggregate differences, we also discovered similar patterns of morphological specification and diversification. Lineage identity plus Notch state govern primary neuronal trajectories, whereas temporal fates diversify terminal elaborations in target-specific manners. In addition, we identified ‘related’ lineages of analogous neuron types produced in similar temporal patterns. Two stem cells even yield identical series of dopaminergic neuron types, but with completely disparate sister neurons. These phenomena suggest that large changes in morphological diversity can be the consequence of relatively small differences in lineage fating. Taken together, this large-scale lineage mapping study reveals that relatively simple rules drive incredible neuronal complexity.

Identification of neuronal lineages in the Drosophila peripheral nervous system with a novel multi-spectral lineage tracing system

SUMMARYElucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multispectral labeling techniques, such as Brainbow, have the potential to identify lineage-related cells in situ. Here, we report Lineage Tracker Bitbow, a “digital” version of Brainbow that greatly expands the color diversity, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools to Drosophila peripheral nervous system, we determined lineage relationship between all neuronal pairs. Based on the refined lineage map, we explored whether distinct cis-regulatory elements are used in controlling the expression of a terminal selector gene in distinct lineage patterns. This study demonstrates LT-Bitbow as an efficient tool for in-situ lineage mapping and its potential in studying molecular mechanisms in the lineage context.

Skin inflammation driven by differentiation of quiescent tissue-resident ILCs into a spectrum of pathogenic effectors

Psoriasis pathology is driven by the type 3 cytokines IL-17 and Il-22, but little is understood about the dynamics that initiate alterations in tissue homeostasis. Here, we use mouse models, single-cell RNA-seq (scRNA-seq), computational inference and cell lineage mapping to show that psoriasis induction reconfigures the functionality of skin-resident ILCs to initiate disease. Tissue-resident ILCs amplified an initial IL-23 trigger and were sufficient, without circulatory ILCs, to drive pathology, indicating that ILC tissue remodeling initiates psoriasis. Skin ILCs expressed type 2 cytokines IL-5 and IL-13 in steady state, but were epigenetically poised to become ILC3-like cells. ScRNA-seq profiles of ILCs from psoriatic and naïve skin of wild type (WT) and Rag1-/- mice form a dense continuum, consistent with this model of fluid ILC states. We inferred biological “topics” underlying these states and their relative importance in each cell with a generative model of latent Dirichlet allocation, showing that ILCs from untreated skin span a spectrum of states, including a naïve/quiescent-like state and one expressing the Cd74 and Il13 but little Il5. Upon disease induction, this spectrum shifts, giving rise to a greater proportion of classical Il5- and Il13- expressing “ILC2s” and a new, mixed ILC2/ILC3-like subset, expressing Il13, Il17, and Il22. Using these key topics, we related the cells through transitions, revealing a quiescence-ILC2-ILC3s state trajectory. We demonstrated this plasticity in vivo, combining an IL-5 fate mouse with IL-17A and IL-22 reporters, validating the transition of IL-5–producing ILC2s to IL-22– and IL-17A–producing cells during disease initiation. Thus, steady-state skin ILCs are actively repressed and cued for a plastic, type 2 response, which, upon induction, morphs into a type 3 response that drives psoriasis. This suggests a general model where specific immune activities are primed in healthy tissue, dynamically adapt to provocations, and left unchecked, drive pathological remodeling.

Defining common principles of gene co-expression refines molecular stratification in cancer

Cancers converge onto shared patterns that arise from constraints placed by the biology of the originating cell lineage and microenvironment on recurrent programs driven by oncogenic events. This structure should be transferable to molecular stratification. We exploit expression data resources and a parsimonious and computationally efficient network analysis method to define consistent expression modules in colon and breast cancer. Comparison between cancer types identifies principles of gene co-expression: cancer hallmarks, functional and structural gene batteries, copy number variation and biology of originating lineage. Mapping outcome data at gene and module level onto these networks generates a detailed interactive resource. Testing the utility of the resulting modules in TCGA data defines specific associations of module expression with mutation state, identifying striking associations such as mast cell gene expression and mutation pattern in breast cancer. These analyses provide evidence for a generalizable framework to enhance molecular stratification in cancer.