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.
Highlights
Analyzing gene expression in multicellular organisms involves a tradeoff between the spatial precision of imaging and the efficiency and comprehensiveness of genomic methods
RNA in situ hybridization (ISH) and antibody staining of fixed samples, or fluorescent imaging of live samples, provides high resolution spatial information for small numbers of genes [1,2,3]
We chose to focus on anterior-posterior (A–P) patterning in the early D. melanogaster embryo, as the system is extremely well-characterized and the geometry of the early embryo lends itself to biologically meaningful physical dissection by simple sectioning along the elongated A–P axis
Summary
Analyzing gene expression in multicellular organisms involves a tradeoff between the spatial precision of imaging and the efficiency and comprehensiveness of genomic methods. As we often recovered more RNA from each embryo than was required to obtain accurate measures of gene expression, we wondered whether we could obtain good data from pieces of individual embryos, and whether we could obtain reliable spatial expression information from such data. To test this possibility, we chose to focus on anterior-posterior (A–P) patterning in the early D. melanogaster embryo, as the system is extremely well-characterized and the geometry of the early embryo lends itself to biologically meaningful physical dissection by simple sectioning along the elongated A–P axis
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