Somitogenesis is a robust mechanism of pattern formation in the development of vertebrates. Although many key factors of somitogenesis such as fgf8a have been characterized, a quantitative analysis of their action remains poorly documented. Such investigations require the control of the concentration, timing and location of the relevant molecules, which can’t be achieved simultaneously by conventional genetic knock-in/out approaches. To fill this gap our lab has developed an optical method combining the use of Gal4-ERT/UAS:fgf8a system and a caged ligand, namely caged-cyclofen. Upon illumination with UV light (at 370nm) or with a two-photon laser beam, cyclofen is released in the illuminated cell(s). It binds to the hormone binding domain of a truncated estrogen receptor (ERT) fused to the Gal4 transcription factor and then releases the Gal4-ERT from the complex it forms with cytoplasmic chaperones. The active Gal4 diffuses to the nucleus, binds to its UAS promoter and turns on the fgf8a gene. This method allows us to over-express fgf8a in live zebrafish in a controlled manner at given time and spatial location.
We have thus analyzed the effect of global over-expression of the fgf8 activated at 70% epiboly and 5 somites stage. The expression of fgf8a was measured using RT-qPCR and its action on somitogenesis was quantitated by time lapse microscopy. While the period of the somitogenetic clock was unaffected by the over-expression of fgf8a, we observed different shortening levels of the somites upon different expression levels and timing of fgf8a over-expression.