Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development.

Transcription factors have diverse roles during embryonic development, combinatorially controlling cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholino oligonucleotides with photoactivatable fluorophores, fluorescence-activated cell sorting and microarray technologies. As a proof of principle, we have dynamically profiled No tail a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies reveal new regulators of notochord development and the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor and demonstrate how optically controlled chemical tools can dissect developmental processes with spatiotemporal precision.

Versatile synthesis and rational design of caged morpholinos.

Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.