Optical control of protein expression and activity at the single cell level: applications to morphogenesis in zebrafish

Living organisms are made of cells that are capable of responding to external signals by modifying their internal state (gene expression or protein phosphorylation patterns) and subsequently their external environment by the release of signaling molecules.  In multicellular organisms in particular, cellular differentiation and signaling is essential for the development of the organism. While many of the key actors of these processes are known (morphogens in development, kinases in signal transduction) much less is known of the quantitative rules that govern their interaction with one another and with other cellular players (such as the type of complexes, rate constants, strength of feedback or feedforward loops, etc).

We initially investigated the development of means to optically control the expression and activity of proteins at the single cell level in a live organism and to use that method to study morphogenesis in zebrafish. The idea is to cage  small soluble biomolecules which  can be released locally with two-photon excitation. We can thus control the activity of proteins and the local development of  the organism.

We have first tested our idea on caged retinoic acid (RA) a potent teratogen. We have shown that the caged compound can passively penetrate a zebrafish embryo with no biological activity.  When uncaged it induces (like RA) major developmental defects. Using two photon excitation in order to release (within a sec) caged RA in a few cells of the retina, we observed a dose dependent increase in the probability of eye malformation observed 15h later. We have discovered that if a second pulse of RA is released more than 5 min. after the first one it has no effect on the development of the eye. We have shown that the kinase p38a is a key element in that fast inhibition of the teratogenic effects of second puff of RA.

Recently we were able to uncage cyclofen in order to control the activity of various proteins (the recombinase Cre and a GFP-nls (see movie)) fused to its receptor (ERT) at the single cell level in a live embryo. These results suggest that single cell control of morphogen gradients is possible in a live organism which should open the way for a precise comparison between morphogenetic models and observations.

Fig.1: Two-photon uncaging of cyclofen in a single cell of a fish retina induces activity of a Cre recombinase that excises a GFP gene and turns on a RFP gene in an appropriate transgenic fish line.

Following is an overnight confocal movie of the development of a zebrafish line which is expressing GFP in rhombomeres 3 and 5 of its hindbrain and a confocal movie of a (nuclear localized) GFP fused to the ERT receptor and activated in a single cell of a zebrafish tail by cyclofen uncaging.