The spatial information associated with gene expression is important for elucidating the context-dependent transcriptional regulation during development. Recently, high-resolution sampling approaches, such as RNA tomography or single-cell RNA-seq combined with fluorescence in situ hybridization (FISH), have provided indirect ways to view global gene expression patterns in three dimensions. Now in situ sequencing technologies, such as fluorescent in situ sequencing (FISSEQ), are attempting to visualize the genetic signature directly in microscope images. Researchers from Cold Spring Harbor Laboratory examine the basic principle of modern in situ and single-cell genetic methods, hurdles in quantifying intrinsic and extrinsic forces that influence cell decision-making, and technological requirements for making a visual map of gene regulation, form, and function. Successfully addressing these challenges will be essential for investigating the functional evolution of regulatory sequences during growth, development, and cancer progression.
Focusing technology development around a central question enables multiple creative approaches necessary to measure specific elements, rather than simply scaling up existing technologies. For example, positional information in developmental biology has been investigated under the assumption of idealized morphogen gradients without considering cellular and environmental variations. If true, any fluctuations in the signal strength due to environmental factors can make precise tissue patterning difficult over a global scale. To understand what makes interpreting positional information robust, our laboratory is developing three distinct in situ sequencing methods capable of measuring single-cell variations, microenvironmental heterogeneity, and cell lineages. In the future, it should be possible to perform selective knockdown of genetic pathways or optogenetic induction of morphogen signaling in vivo and quantify how intrinsic, extrinsic, and lineage-specific factors drive location-specific cell fate commitment and differentiation.