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Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.

The connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Krüppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

The thymus is a primary lymphoid organ that is essential for the establishment of adaptive immunity through generation of immunocompetent T cells. In response to various stress signals, the thymus undergoes acute but reversible involution. However, the mechanisms governing its recovery are incompletely understood. Here, we used a dexamethasone-induced acute thymic involution mouse model to investigate how thymic hematopoietic cells (excluding T cells) contribute to thymic regeneration. scRNA-seq analysis revealed marked transcriptional and cellular changes in various thymic populations and highlighted thymus-resident innate lymphoid cells type 2 (ILC2) as a key cell type involved in the response to damage. We identified that ILC2 are activated by the alarmins IL-25 and IL-33 produced in response to tissue damage by thymic tuft cells and fibroblasts, respectively. Moreover, using mouse models deficient in either tuft cells and/or IL-33, we found that these alarmins are required for effective thymus regeneration after dexamethasone-induced damage. We also demonstrate that upon their damage-dependent activation, thymic ILC2 produce several effector molecules linked to tissue regeneration, such as amphiregulin and IL-13, which in turn promote thymic epithelial cell differentiation. Collectively, our study elucidates a previously undescribed role for thymic tuft cells and fibroblasts in thymus regeneration through activation of the type 2 immune response. Editor's summary: The thymus undergoes transient but reversible involution in response to stress stimuli, but the molecular mechanisms mediating thymic regeneration after involution are not well defined. Nevo et al. performed scRNA-seq on the thymic non–T cell compartment after dexamethasone-induced acute thymic involution (ATI) in mice. Dexamethasone-induced ATI promoted a type 2 immune response driven by thymic resident ILC2s that were activated in response to tissue damage by IL-25 produced by thymic tuft cells and IL-33 produced by fibroblasts. ILC2s facilitated tissue regeneration by producing effector molecules including AREG, IL-13, CSF2, and IL-5, which promoted mTEC differentiation. Together, these findings identify a thymic tuft cell–fibroblast–ILC2 axis required for thymic regeneration after ATI. —Hannah Isles [ABSTRACT FROM AUTHOR]

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB γ‐lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell‐autonomously required in γ‐neurons for their developmental regrowth into the distal γ4/5 zones, where both Dpr12 and its interacting protein, DIP‐δ, are enriched. DIP‐δ functions in a subset of dopaminergic neurons that wire with γ‐neurons within the γ4/5 zone. During metamorphosis, these dopaminergic projections arrive to the γ4/5 zone prior to γ‐axons, suggesting that γ‐axons extend through a prepatterned region. Thus, Dpr12/DIP‐δ transneuronal interaction is required for γ4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution. Synopsis: Precise circuit formation is crucial for neuronal function. Here, we identify trans‐synaptic interactions of Immunoglobulin Superfamily proteins as mediators of compartmentalized dopaminergic innervation in the Drosophila mushroom body. The IgSF protein Dpr12 is cell‐autonomously required in γ‐Kenyon cells for their complete developmental regrowth.Dpr12 and its interacting protein DIP‐δ are both enriched in the MB γ4–5 zones, defined by compartmentalized innervations of dopaminergic and MB output neurons.DIP‐δ functions within a cluster of dopaminergic neurons that innervates the γ4–5 zones.The Dpr12‐DIP‐δ interaction is required for the formation of the γ4–5 zones.DIP‐δ is required and sufficient for the localization of Dpr12, in trans. [ABSTRACT FROM AUTHOR]