Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early<i>Drosophila</i>embryo

Ho, Emily K; Oatman, Harrison R; McFann, Sarah; Liu, Yang; Johnson, Heath E.; Shvartsman, Stanislav Y.; Toettcher, Jared E.

doi:10.1101/2023.03.09.531972

Cited by 2 publications

(2 citation statements)

References 52 publications

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“…LOV domains make up the core of most non-ion-channel optogenetic systems [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] , presumably because they are small, reliable, and do not need exogenous chromophores. Interest in LOV domains is reflected in multiple diversification campaigns 34,[54][55][56] .…”

Section: )mentioning

confidence: 99%

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution isin vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss ofYOR1makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

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Section: )mentioning

confidence: 99%

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Gligorovski,

Labagnara,

Rahi

2024

Preprint

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“…In developmental biology, optogeneHc strategies have been applied most extensively to invesHgate terminal paNerning via the Ras/ERK signaling pathway in the early Drosophila embryo 14,[41][42][43] . These approaches have now been applied to several morphogen pathways 44,45,46 as well as to vertebrate embryos 47,21,48 ; however, pracHcal challenges have prevented widespread adopHon.…”

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confidence: 99%

Optogenetic control of Nodal signaling patterns

McNamara,

Jia,

Guyer

et al. 2024

Preprint

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A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light-sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.

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Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the earlyDrosophilaembryo

Cited by 2 publications

References 52 publications

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

Optogenetic control of Nodal signaling patterns

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