van Buggenum, J. A. G. | Search bioRxiv

bioRxiv2018

Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogenenous cellular responses to environmental signals at the mRNA and phospho-proteome level.

genomics↗

bioRxiv2018

Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program.

AbstractEpidermal homeostasis requires balanced and coordinated adult stem cell renewal and differentiation. These processes are controlled by both extracellular signaling and by cell intrinsic transcription regulatory networks, yet how these control mechanisms are integrated to achieve this unclear. Here, we developed single-cell ID-seq and measured 69 antibody-DNA conjugates (including 34 phospho-specific epitopes) to study the activation state of signaling pathways during epidermal differentiation at the single-cell level. Computational pseudo-timing inference revealed activation of the JAK-STAT, WNT and BMP pathways along the epidermal differentiation trajectory. During differentiation, cells start producing BMP2 ligands and activate the canonical intracellular effectors SMAD1/5/9. Mechanistically, the BMP pathway is responsible for directly activating a specific transcription program that includes the key differentiation transcription factors MAF and MAFB to allow terminal differentiation. We propose that incorporating autocrine signaling pathway activation into a transcription regulatory network enables regional coordination of transcription programs during epidermal differentiation.

developmental biology↗