Sequencing mRNA from cryo-sliced Drosophila embryos to determine genome-wide spatial patterns of gene expression. Peter A. Combs¹, Michael B. Eisen^2,3. 1) Biophysics Graduate Group, University of California, Berkeley, CA; 2) Department of Molecular and Cell Biology, University of California, Berkeley, CA; 3) Howard Hughes Medical Institute, University of California, Berkeley, CA.

   Spatially patterned gene expression underlies animal development, yet methods do not yet exist for the genome-wide determination of spatial patterns of gene expression. Fluorescent imaging of transcripts and proteins is the gold-standard, but is relatively slow and expensive to expand to an entire genome, even when highly automated. In contrast, sequencing is fast and genome-wide, but discards spatial information by operating on homogenized tissues. Here we developed a method of rapidly determining genome-wide spatial patterns of gene expression to identify genes with previously undescribed spatial expression patterns, and to investigate the effects of mutants and other perturbations on patterned gene expression. To do this, we developed methods to sequence mRNA from single 60m cryosections of Drosophila melanogaster embryos at the blastoderm stage. We identify numerous maternally deposited genes with spatial patterns, including many not yet screened in systematic in situ based approaches. The majority of these are localized to pole cells, although we also observe anterior localization. We also detected spatially varying usage of individual exons in transcriptional regulators, which could not have been identified in previous sequencing analyses. Finally, we compared wild-type embryos with bicoid dosage mutants, allowing us to determine concentration-dependent transcriptional responses of hundreds of Bicoid target genes simultaneously. Overall, our results fill in key gaps in knowledge that spatially homogenized approaches cannot address, and demonstrate the power of combining sectioning or anatomical dissection to provide missing spatial information to sequencing-based genomic studies.