A live-cell biosensor of in vivo receptor tyrosine kinase activity reveals feedback regulation of a developmental gradient

Emily K Ho; Rebecca P Kim-Yip; Alison G Simpkins; Payam E Farahani; Harrison R Oatman; Eszter Posfai; Stanislav Y Shvartsman; Jared E Toettcher

doi:10.1016/j.celrep.2025.115930

A live-cell biosensor of in vivo receptor tyrosine kinase activity reveals feedback regulation of a developmental gradient

Cell Rep. 2025 Jun 27;44(7):115930. doi: 10.1016/j.celrep.2025.115930. Online ahead of print.

Authors

Emily K Ho¹, Rebecca P Kim-Yip², Alison G Simpkins³, Payam E Farahani⁴, Harrison R Oatman³, Eszter Posfai², Stanislav Y Shvartsman⁵, Jared E Toettcher⁶

Affiliations

¹ Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Electronic address: ekho@princeton.edu.
² Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
³ Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
⁴ Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA.
⁵ Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA; Center for Computational Biology, Flatiron Institute - Simons Foundation, New York, NY 10010, USA.
⁶ Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ 08544, USA. Electronic address: toettcher@princeton.edu.

PMID: 40581929
DOI: 10.1016/j.celrep.2025.115930

Abstract

A lack of tools for detecting receptor activity in vivo has limited our ability to fully explore receptor-level control of developmental patterning. Here, we extend phospho-tyrosine tag (pYtag) biosensors to visualize endogenous receptor tyrosine kinase (RTK) activity in Drosophila. We build biosensors for three RTKs that function across developmental stages and tissues. By characterizing Torso::pYtag during embryonic terminal patterning, we find that Torso activity differs from downstream extracellular signal-regulated kinase (ERK) activity in two surprising ways: Torso activity is narrowly restricted to the poles but produces a broader gradient of ERK and decreases over developmental time, while ERK activity is sustained, an effect mediated by ERK pathway-dependent negative feedback. Our results suggest that a narrow domain of Torso activity, tuned in amplitude by negative feedback, locally activates signaling effectors, which diffuse through the syncytial embryo to form the ERK gradient. Altogether, the results of this work highlight the usefulness of pYtags for investigating receptor-level regulation of developmental patterning.

Keywords: CP: Cell biology; CP: Developmental biology; Drosophila development; ERK signaling; biosensor; receptor tyrosine kinases.