Embryo-scale single-cell chemical transcriptomics reveals dependencies between cell types and signaling pathways

Abstract

Organogenesis is a fast and coordinated process that is highly conserved across vertebrates. However, we lack a detailed map of how each developing cell type in each organ depends on the developmental signaling pathways. Conventional gene knockouts cannot target an entire signaling pathway or have severe early phenotypes, making it challenging to study regulation of organogenesis. To fill this gap in knowledge, we applied highly multiplexed, scalable and temporally controlled in vivo chemical perturbations to dissect individual signaling pathways’ (BMP, FGF, Notch, RA, Shh, TGFβ & Wnt) roles in organogenesis. Here we present the transcriptional data of 711,997 cells from 288 chemically perturbed zebrafish embryos, spanning multiple time points (36, 48 and 72 hours post fertilization (hpf)) in organogenesis. The high degree of replication in our approach, which independently profiles multiple embryos per condition with DNA barcodes using Sci-Plex, allows us to statistically calculate the variance in cell type abundance and gene expression, embryo-wide and detect perturbation-dependent changes, even in very rare cell types. With this approach, I define a list of cell types that utilize each signaling pathway as well as undergo cross-regulation from other signaling pathways. I demonstrate that differential global gene expression analysis can identify novel signaling pathway regulation of transcription factors (TFs) in select cell types. I characterize novel signaling pathway regulation of multiple cell types within the pectoral fin mesoderm, a very rare tissue comprising less than 1% of the embryo. Shh is known to positively regulate pectoral fin mesoderm and cartilage, however, we show Shh also negatively regulates cleithrum size. In addition to comprehensively characterizing signaling pathway regulation of the pectoral fin over time, I constructed the highest-resolution trajectory to date of the developing pectoral fin mesoderm and identified two previously uncharacterized lateral plate mesoderm (LPM)-derived cell types as well as multiple novel gene markers of these LPM-derived pectoral fin cell types. Transcriptional and spatial analysis characterized one of the unknown cell types as distal mesenchyme, which was previously mischaracterized as an ectoderm-derivative, and the other unknown cell type as a tenocyte population that we show lie in between the muscle and the cartilage, presumably serving a functional role of anchoring the two cell types together. Taken together, our data provides a valuable resource for understanding the development of paired appendages and the comprehensive roles of signaling pathways in regulating vertebrate organogenesis in embryos as a whole.In this dissertation, I will first introduce how essential signaling pathways are in regulating embryonic development and how previous techniques to study their roles were neither comprehensive nor high-throughput, limiting discovery of novel biology. I will introduce how single-cell technologies can fill the need to profile embryos in an unbiased, comprehensive and high-throughput way. Using zebrafish pectoral fins as an example, I show how we can use this approach to develop models of signaling pathway regulation within individual tissue lineages.