Science Spotlight - zHORSE
Precision in gene regulation with zHORSE: No more horsing around
By Anna Vinken
Optogenetic technologies promise a way forward for better control of gene expression: light is non-invasive, precise, and ideally suited to transparent zebrafish embryos. Still, no existing optogenetic system fully met the demands of modern zebrafish research. Several innovative optogenetic systems, including iCreV, TAEL, and GAVPO, have advanced zebrafish research by offering improved control of gene expression4-6 (for an example, see the other Science Spotlight article in this issue of the News Splash). However, none have yet fulfilled all the desired criteria for a truly versatile in vivo tool with exquisite temporal and spatial resolution up to the single-cell level, consistent functionality throughout development, ease of use without sophisticated infrastructure, and compatibility with existing transgenic effector strains.
Recognizing these limitations, Varady et al. have developed a zebrafish heat-shock-inducible optogenetic recombinase expression (zHORSE). This novel dual-gated gene expression system overcomes previous challenges by integrating heat and light control to achieve precise, reversible, and low-background gene activation. By combining a heat-shock promoter with a light-activated transcription factor, zHORSE requires a brief heat pulse followed by targeted blue-light illumination to activate gene expression. This dual requirement effectively eliminates background activity, allowing reversible and highly localized induction at single-cell and even subcellular precision. This combination transforms a leaky, single-trigger system into a reversible, tightly controlled platform for precise gene expression. Remarkably, the system performs consistently from gastrulation through late embryogenesis and can be used with standard lab equipment, making it both powerful and practical.
The first author Adam Varady highlights the motivation behind the work: “Obviously, as cancer researchers, we mainly wanted it to induce oncogenes or other genetic manipulations. But as we’ve shown, it might also be interesting for development. You could use it for global induction, or for lineage tracing.”
Functionally, zHORSE opens the door to experiments that were previously aspirational: probing the exact timing of signaling pathway function, testing gene sufficiency in single cells during fate decisions, and tracing how individual cells behave in response to targeted genetic perturbations — all with near-surgical precision. For example, the authors used zHORSE for lineage tracing of caudal fin progenitor cells, revealing dynamic contributions to fin development. They also demonstrated optogenetic tissue manipulation by inducing oncogene expression, specifically targeted activation of the EWS::FLI1 fusion oncogene, which triggered ectopic fin formation. This manipulation further revealed that FGF signaling acts downstream of EWS::FLI1, illustrating how zHORSE can dissect complex genetic pathways in vivo with temporal and spatial control.
“I was just fascinated by the new optogenetic field when it first developed.” recalls PI Martin Distel on how the idea for this tool began. “And of course, for zebrafish being a transparent model organism, it was pretty obvious that this needs some applications in zebrafish too.”
Of course, some challenges and limitations remain. zHORSE cannot be used before about 9 hours post-fertilization due to heat-shock sensitivity, and careful shielding from ambient light—including monitors and phone screens—is needed to avoid unintended activation. The need for localized blue-light and heat pulses may complicate some experiments, while future improvements such as red-light filters, two-photon activation, and tissue-specific effector lines could enhance precision and usability.
“One thing we would have loved to try, and will now, is activation with a two-photon system,” says Distel, reflecting on next steps. “We saw some off-target expression in about half of the cases, and our idea is that this comes from scattering of photons. If you could go in with a two-photon system where the wavelength of the scattering photons would not really activate the system, we might get rid of that.” Beyond technical refinements, Distel also sees enormous potential for creative applications. “We think of zHORSE as a body-engineering tool. You could, for example, induce transcription factors in very specific sites and see what really happens if you start an organ program or an appendage program there. I think there are a ton of exciting opportunities, and we hope the community use it.”
zHORSE represents a major step toward a long-standing goal in developmental genetics: controlling gene expression in exactly the right cell at the right place and time — and doing so with ease.
Adam Varady completed his PhD in Martin Distel’s lab at the St. Anna Children’s Cancer Research Institute in Vienna, Austria. His research focused on using light-controlled proteins and chemical compounds to create new experimental approaches for cancer modeling, tumor therapy, developmental biology and lineage tracing. These methods aimed to provide more precise tools for studying complex biological processes with high spatial resolution and contribute to a broader understanding of disease mechanisms.
Dr. Martin Distel has recently moved to the University of Utah where he is an Associate Professor in the department of Pediatrics and an Investigator at the Huntsman Cancer Institute. His research focuses on pediatric cancers, with a particular emphasis on pediatric sarcomas. His lab develops zebrafish models for these cancers using xenotransplantation and genetic modeling strategies. His team applies these models to investigate molecular mechanisms driving the disease and to develop novel therapeutic strategies.
Anna Vinken is a Master’s student in Cancer, Stem Cell, and Developmental Biology at Utrecht University. She is currently completing her internship in the Barakat lab, part of the Clinical Genetics Department at Erasmus MC, under the supervision of Leslie Sanderson. During this internship, she was first introduced to zebrafish, an experience that sparked her fascination for this model organism. Her current research focuses on disease modeling, particularly in the context of neurodevelopmental disorders. Outside of the lab, Anna enjoys caring for her many houseplants, maintaining a collection so extensive that she keeps more than one plant per square meter in her home.
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