Precise gene expression measurement has been fundamental to developing an advanced understanding of the roles of biological networks in health and disease. To enable detection of expression signatures specific to individual cells we developed PLAYR (Proximity Ligation Assay for RNA). PLAYR enables highly multiplexed quantification of transcripts in single cells by flow- and mass-cytometry and is compatible with standard antibody staining of proteins. With mass cytometry, this currently enables simultaneous quantification of more than 40 different mRNAs and proteins. The technology was demonstrated in primary cells to be capable of quantifying multiple gene expression transcripts while the identity and the functional state of each analyzed cell was defined based on the expression of other transcripts or proteins. PLAYR now enables high throughput deep phenotyping of cells to readily expand beyond protein epitopes to include RNA expression, thereby opening a new venue on the characterization of cellular metabolism.

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun–mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

Summary Hematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout an animal’s life 1 . While there are comprehensive studies on vertebrate HSC self-renewal, differentiation, physiological regulation and niche occupation, relatively little is known about their evolutionary origin and their niches. Here we study the hematopoietic system of Botryllus schlosseri , a colonial tunicate that has vasculature, circulating blood cells, and interesting stem cell biology and immunity characteristics 2 – 8 . Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other 3 , 4 , 7 . Using flow-cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, and an HSC niche, and demonstrated that self-recognition inhibits allospecific cytotoxic reactions. Our study reveals that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and these results also suggest that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.