title: Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish.
abstract: To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation.
authors.affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA. tallafuss@uoneuro.uoregon.edu
authors.forename: Alexandra
authors.initials: A
authors.lastname: Tallafuss
authors.forename: Dan
authors.initials: D
authors.lastname: Gibson
authors.forename: Paul
authors.initials: P
authors.lastname: Morcos
authors.forename: Yongfu
authors.initials: Y
authors.lastname: Li
authors.forename: Steve
authors.initials: S
authors.lastname: Seredick
authors.forename: Judith
authors.initials: J
authors.lastname: Eisen
authors.forename: Philip
authors.initials: P
authors.lastname: Washbourne
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D005326:Fetal Proteins; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000528:radiation effects*; D007150:Immunohistochemistry; D060172:Morpholinos / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D009046:Motor Neurons / Q000378:metabolism; D009432:Neural Crest / Q000166:cytology / Q000378:metabolism; D009672:Notochord / Q000166:cytology / Q000196:embryology; D055757:SOXE Transcription Factors / Q000378:metabolism*; D020825:T-Box Domain Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism*; D000098908:Brachyury Protein
chemical_list: D004268:DNA-Binding Proteins; D005326:Fetal Proteins; D060172:Morpholinos; D055757:SOXE Transcription Factors; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000098908:Brachyury Protein; C542436:Gal4 protein, zebrafish; C442229:sox10 protein, zebrafish