Manipulate, Watch, & Learn: OptoSOS sheds light on how the fly earns its stripes

Summary by Cat Rogers: Ho, E.K., Oatman, H.R., McFann, S.E., Yang, L., Johnson, H.E., Shvartsman, S.Y., & Toettcher, J.E. “Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo” bioRxiv, doi:10.1101/2023.03.09.531972 (2023).

Image credit: Midjourney

The Brachyenteron (byn) gene is expressed in a stripe in the posterior of the Drosophila embryo, a model organism known for its distinct gene expression stripes. Byn expression is regulated by the Ras/ERK signaling pathway. Ras/ERK signaling inactivates a repressor of two transcription factors that influence byn expression: Tailless (Tll), which activates byn, and Huckebein (Hkb), which inhibits byn. The Ras/ERK signaling effector, dpERK, forms a gradient with high concentrations at the terminal region and low concentrations at more interior positions. tll expression spans the entire dpERK gradient while hkb expression is limited to regions with higher dpERK levels. This regulatory circuit seems convoluted; it is counterintuitive that ERK would activate two factors with opposite effects on byn expression. So, what is going on?

byn’s expression domain is dynamic.1,2 It spans the entire dpERK gradient briefly before expression is lost in more terminal positions, ultimately creating the byn stripe. To uncover the mechanisms underlying dynamic byn stripe formation, Ho et al. 3 use an optogenetic activator of Ras/ERK signaling, OptoSOS4,5, together with a live-cell readout of gene expression, the MS2/MCP system6,7, to tag nascent transcripts of interest and indicate active gene expression. Combining these methods allows for manipulation and visualization of byn network dynamics.

First, Ho et al. looked at how byn responds to ectopic ERK activation. Since OptoSOS activity is light intensity-dependent,5  they were able to simulate different positions along the endogenous dpERK gradient by activating different levels of signaling. The high-light / high-signaling level condition produced a transient pulse of byn expression, while the low-light / low-signaling condition produced sustained byn expression. This recapitulates the dynamic endogenous byn expression domain: In regions with high dpERK levels, byn is expressed at early time points but turns off at later times, while in regions with lower dpERK levels, byn expression is sustained.

Next, the authors investigated how upstream regulators of byn—the activator tll and inhibitor hkb—respond to the same optogenetic manipulations. In both high- and low-light conditions, tll was activated immediately and thus does not appear to respond differently to different dpERK levels. In contrast, hkb activity was intensity-dependent: in high-light conditions, there is a short delay of hkb transcription relative to tll, whereas in low-light conditions, hkb transcription was delayed much longer. Thus, at more terminal positions in the embryo, high dpERK leads to transient byn expression, while at more interior positions low dpERK leads to sustained byn expression. This leaves a byn stripe at more interior positions. These results suggest that different levels of the dpERK gradient are decoded as a differential time delay of hkb expression. 

Using OptoSOS combined with rapid live-cell transcription readouts, Ho et al. illuminate how a complex incoherent feed-forward loop earns the fly a byn stripe. This study showcases how optogenetics can allow researchers to manipulate signaling inputs and observe the immediate, dynamic gene expression outputs. More studies like this, in Drosophila and other organisms, will help deepen our understanding of how well-studied and enigmatic gene networks alike orchestrate development.

1. Keenan, S. E. et al. Dynamics of Drosophila endoderm specification. Proc Natl Acad Sci U S A 119, e2112892119, doi:10.1073/pnas.2112892119 (2022).

2. Kispert, A., Herrmann, B. G., Leptin, M. & Reuter, R. Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta. Genes & Development (1994).

3. Ho, E. K. et al. Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. bioRxiv, doi:10.1101/2023.03.09.531972 (2023).

4. Toettcher, J. E., Weiner, O. D. & Lim, W. A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422-1434, doi:10.1016/j.cell.2013.11.004 (2013).

5. Johnson, H. E. et al. The Spatiotemporal Limits of Developmental Erk Signaling. Dev Cell 40, 185-192, doi:10.1016/j.devcel.2016.12.002 (2017).

6. Bertrand, E. et al. Localization of ASH1 mRNA Particles in Living Yeast. Molecular Cell 2 (1998).

7. Forrest, K. M. & Gavis, E. R. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr Biol 13, 1159-1168, doi:10.1016/s0960-9822(03)00451-2 (2003).