Genome-wide RNA Tomography in the Zebrafish Embryo

Abstract

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos

“…Relative levels of gene expression are often studied by RNA-sequencing approaches. When performed at the cellular level, however, this technique only detects the ∼10% most abundant transcripts and is thus rather insensitive (Grün et al, 2014;Junker et al, 2014a;Satija et al, 2015). Furthermore, neither technique provides subcellular resolution.…”

“…In developmental biology, for instance, it provides valuable information on the role of differential gene expression in determining cell fates (Junker et al, 2014a;Satija et al, 2015;Thisse and Thisse, 2008;Tomancak et al, 2007). Spatial patterns of gene expression have historically been studied by RNA in situ hybridization, but this technique is generally not quantitative (Gross-Thebing et al, 2014;Thisse and Thisse, 2008;Tomancak et al, 2007).…”

Avian transcriptomics: opportunities and challenges

“…Maintaining spatial information on gene expression of single cells or subpopulations of cells in tissues could help us to better understand how different cells function and are regulated, where they are localised and how they interact in complex tissues (Crosetto et al 2015). Some techniques can be used in combination with RNA-seq such as laser-capture microdissection, where single cells or subpopulations of cells can be harvested from tissue samples and used for downstream analysis (Espina et al 2006); microtomy sequencing, where RNA is extracted from thin cryosections (Junker et al 2014); or spatial transcriptomics where tissues are positioned on an array with spatially barcoded primers, which allow for two-dimensional positional information to be taken into account in the analysis (Ståhl et al 2016). These technologies offer new possibilities to learn more about avian biology, in particular within areas such as neurobiology and immunology.…”

Blood stem cells: from beginning to end

“…One of the important pathways identified in the hemogenic endothelium was Hippo signaling, which may be an important regulator in the early stages of hematopoietic differentiation. A relatively new and innovative approach to understanding gene expression at the four-dimensional level -that is, through both space and time -is 'Tomo-seq', a genome-wide RNA tomography approach introduced by Laurent Yvernogeau, a postdoctoral fellow in Catherine Robin's Lab (Junker et al, 2014). He collected and sequenced sequential sections of whole aortas (cut along the anteriorto-posterior axis) and thick transversal embryo slices (cut along the ventral-to-dorsal axis) from various embryo species isolated at different developmental stages: before, during and after cluster/HSC emergence.…”