scRNA-sequencing in chick suggests a probabilistic model for cell fate allocation at the neural plate border.

The vertebrate 'neural plate border' is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate, neural crest, placodes and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules, allowing us to model the order of molecular events which take place as ectodermal fates segregate. Furthermore, we find that genes previously classified as neural plate border 'specifiers' typically exhibit dynamic expression patterns and are enriched in either neural, neural crest or placodal fates, revealing that the neural plate border should be seen as a heterogeneous ectodermal territory and not a discrete transitional transcriptional state. Analysis of neural, neural crest and placodal markers reveals that individual NPB cells co-express competing transcriptional programmes suggesting that their ultimate identify is not yet fixed. This population of 'border located undecided progenitors' (BLUPs) gradually diminishes as cell fate decisions take place. Considering our findings, we propose a probabilistic model for cell fate choice at the neural plate border. Our data suggest that the probability of a progenitor's daughters to contribute to a given ectodermal derivative is related to the balance of competing transcriptional programmes, which in turn are regulated by the spatiotemporal position of a progenitor.

Employing core regulatory circuits to define cell identity.

The interplay between extrinsic signaling and downstream gene networks controls the establishment of cell identity during development and its maintenance in adult life. Advances in next-generation sequencing and single-cell technologies have revealed additional layers of complexity in cell identity. Here, we review our current understanding of transcription factor (TF) networks as key determinants of cell identity. We discuss the concept of the core regulatory circuit as a set of TFs and interacting factors that together define the gene expression profile of the cell. We propose the core regulatory circuit as a comprehensive conceptual framework for defining cellular identity and discuss its connections to cell function in different contexts.

ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

An integrated pipeline for high-throughput screening and profiling of spheroids using simple live image analysis of frame to frame variations.

High-throughput imaging methods can be applied to relevant cell culture models, fostering their use in research and translational applications. Improvements in microscopy, computational capabilities and data analysis have enabled high-throughput, high-content approaches from endpoint 2D microscopy images. Nonetheless, trade-offs in acquisition, computation and storage between content and throughput remain, in particular when cells and cell structures are imaged in 3D. Moreover, live 3D phase contrast microscopy images are not often amenable to analysis because of the high level of background noise. Cultures of Human induced pluripotent stem cells (hiPSC) offer unprecedented scope to profile and screen conditions affecting cell fate decisions, self-organisation and early embryonic development. However, quantifying changes in the morphology or function of cell structures derived from hiPSCs over time presents significant challenges. Here, we report a novel method based on the analysis of live phase contrast microscopy images of hiPSC spheroids. We compare self-renewing versus differentiating media conditions, which give rise to spheroids with distinct morphologies; round versus branched, respectively. These cell structures are segmented from 2D projections and analysed based on frame-to-frame variations. Importantly, a tailored convolutional neural network is trained and applied to predict culture conditions from time-frame images. We compare our results with more classic and involved endpoint 3D confocal microscopy and propose that such approaches can complement spheroid-based assays developed for the purpose of screening and profiling. This workflow can be realistically implemented in laboratories using imaging-based high-throughput methods for regenerative medicine and drug discovery.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

γ-TuRC Heterogeneity Revealed by Analysis of Mozart1.

Microtubules are essential for various cell processes [1] and are nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs) at various microtubule organizing centers (MTOCs), including centrosomes [2-6]. Recruitment of γ-TuRCs to different MTOCs at different times influences microtubule array formation, but how this is regulated remains an open question. It also remains unclear whether all γ-TuRCs within the same organism have the same composition and how any potential heterogeneity might influence γ-TuRC recruitment. MOZART1 (Mzt1) was recently identified as a γ-TuRC component [7, 8] and is conserved in nearly all eukaryotes [6, 9]. Mzt1 has so far been studied in cultured human cells, yeast, and plants; its absence leads to failures in γ-TuRC recruitment and cell division, resulting in cell death [7, 9-15]. Mzt1 is small (∼8.5 kDa), binds directly to core γ-TuRC components [9, 10, 14, 15], and appears to mediate the interaction between γ-TuRCs and proteins that tether γ-TuRCs to MTOCs [9, 15]. Here, we use Drosophila to investigate the function of Mzt1 in a multicellular animal for the first time. Surprisingly, we find that Drosophila Mzt1 is expressed only in the testes and is present in γ-TuRCs recruited to basal bodies, but not to mitochondria, in developing sperm cells. mzt1 mutants are viable but have defects in basal body positioning and γ-TuRC recruitment to centriole adjuncts; sperm formation is affected and mutants display a rapid age-dependent decline in sperm motility and male fertility. Our results reveal that tissue-specific and MTOC-specific γ-TuRC heterogeneity exist in Drosophila and highlight the complexity of γ-TuRC recruitment in a multicellular animal.