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Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal–ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo., Author summary Much of what we know about biological processes has come from the analysis of mutants whose loss-of-function phenotypes provide insight into their normal functions. However, for genes that are required for viability and which have multiple functions in the life of a cell or organism one can only observe mutant phenotypes produced up to the time of death. Normal functions performed in wild-type individuals later than the time of death of mutants cannot be observed. In one approach to overcoming this limitation, a class of peptide degradation signals (degrons) have been developed, which when fused to proteins-of-interest, can target those proteins for degradation in response to various stimuli (temperature, chemical agents, co-expressed proteins, or light). Here we describe a new inducible degron (the photo-N-degron or PND), which when fused to the N-terminus of a protein, can induce N-end rule-mediated degradation in response to blue-light illumination and have validated its use in both yeast and Drosophila embryos. Moreover, using the Drosophila embryonic patterning protein Cactus, we show that like the PND, the previously-described B-LID domain, but not the previously-described photosensitive degron (psd), can produce detectable light-inducible phenotypes in Drosophila embryos that are consistent with the role of Cactus in dorsal-ventral patterning.