Biology subjects

Oatman, H. R.

Publications and source records attributed to Oatman, H. R..

3 recordsLinked to original sources

bioRxiv2025

PyCLM: programming-free, closed-loop microscopy for real-time measurement, segmentation, and optogenetic stimulation

In cell biology, optical techniques are increasingly used to measure cells internal states (biosensors) and to stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a pre-determined stimulus pattern is applied to cells, biosensors are measured over time, and the resulting data is processed off-line. With the advent of machine learning for segmentation and tracking, it becomes possible to envision closed-loop experiments where real-time information about cells positions and states are used to dynamically determine optogenetic stimuli to alter or control their behavior. Here, we develop PyCLM, a Python-based suite of tools to enable real-time measurement, image segmentation, and optogenetic control of thousands of cells per experiment. PyCLM is designed to be as simple for the end user as possible, and multipoint experiments can be set up that combine a wide variety of imaging, image processing, and stimulation modalities without any programming. We showcase PyCLM on diverse applications: studying the effect of epidermal growth factor receptor activity waves on epithelial tissue movement, simultaneously stimulating [~]1,000 single cells to guide tissue flows, and performing real-time feedback control of cell-to-cell fluorescence heterogeneity. This tool will enable the next generation of dynamic experiments to probe cell and tissue properties, and provides a first step toward precise control of cell states at the tissue scale.

systems biology↗

bioRxiv2025

In vivo measurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient

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 a new class of biosensors for receptor tyrosine kinase (RTK) activity, the pYtag system, to visualize endogenous RTK activity in Drosophila. We build biosensors for three Drosophila RTKs that function across developmental stages and tissues. By characterizing Torso::pYtag during terminal patterning in the early embryo, we find that Torso activity differs from downstream ERK activity in two surprising ways: Torso activity is narrowly restricted to the poles but produces a broader gradient of ERK, and Torso activity decreases over developmental time while ERK activity is sustained. This decrease in Torso activity is driven by ERK pathway-dependent negative feedback. Our results suggest an updated model of terminal patterning where 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, this work highlights the usefulness of pYtags for investigating receptor-level regulation of developmental patterning.

developmental biology↗

bioRxiv2023

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

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be blurred through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

developmental biology↗