Sequencing mRNA from Cryo-Sliced Drosophila Embryos to Determine Genome-Wide Spatial Patterns of Gene Expression

Abstract

Complex spatial and temporal patterns of gene expression underlie embryo differentiation, yet methods do not yet exist for the efficient genome-wide determination of spatial expression patterns during development. In situ imaging of transcripts and proteins is the gold-standard, but it is difficult and time consuming to apply to an entire genome, even when highly automated. Sequencing, in contrast, is fast and genome-wide, but is generally applied to homogenized tissues, thereby discarding spatial information. To take advantage of the efficiency and comprehensiveness of sequencing while retaining spatial information, we cryosectioned individual blastoderm stage Drosophila melanogaster embryos along the anterior-posterior axis and developed methods to reliably sequence the mRNA isolated from each 25 µm slice. The spatial patterns of gene expression we infer closely match patterns previously determined by in situ hybridization and microscopy. We applied this method to generate a genome-wide timecourse of spatial gene expression from shortly after fertilization through gastrulation. We identified numerous genes with spatial patterns that have not yet been described in the several ongoing systematic in situ based projects. This simple experiment demonstrates the potential for combining careful anatomical dissection with high-throughput sequencing to obtain spatially resolved gene expression on a genome-wide scale.

Comparing mRNA levels using in situ hybridization of a target gene and co-stain

“…In the interim, it is useful to bring quantitation to standard spatially resolved techniques, such as in situ hybridization; that was our goal with this work. Improvements are also occurring from the other direction: recent work has reduced the sample size required for RNA-seq to single embryo [33] and sub-embryo sizes [34]. Because quantitative, spatially resolved data is a requirement for modeling the behavior of developmental networks, we are optimistic that the field will continue to press forward on this problem.…”

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics

“…Visualization and analysis of gene expression in tissue sections by spatial transcriptomics Patrik L. Ståhl, 1,2 * Fredrik Salmén, 2 * Sanja Vickovic, 2 † Anna Lundmark, 2,3 † José Fernández Navarro, 1,2 Jens Magnusson, 1 Stefania Giacomello, 2 Michaela Asp, 2 Jakub O. Westholm, 4 Mikael Huss, 4 Annelie Mollbrink, 2 Sten Linnarsson, 5 Simone Codeluppi, 5,6 Åke Borg, 7 Fredrik Pontén, 8 Paul Igor Costea, 2 Pelin Sahlén, 2 Jan Mulder, 9 Olaf Bergmann, 1 Joakim Lundeberg, 2 ‡ Jonas Frisén 1…”

Dense Bicoid hubs accentuate binding along the morphogen gradient

“…To test whether BCD is binding with specificity in the posterior embryo, we analyzed its binding profiles in a spatially segregated manner (Combs and Eisen 2013) by comparing ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) profiles derived from individually dissected posterior thirds of embryos with previously published data from whole embryos (Bradley et al 2010). Our analysis reveals that BCD indeed binds to known targets in the posterior but with increased relative enrichment at specific enhancer elements (Fig.…”