Understanding how signalling proteins act in space and time is central to developmental biology. The Hippo pathway and its transcriptional effector Yap1 translate mechanical and chemical cues into transcriptional programs during embryogenesis and regeneration. To interrogate these dynamic processes in vivo, this study developed an experimental tool to both visualise and manipulate endogenous Yap1 in medaka.
The authors aimed to combine endogenous fluorescent tagging with an inducible degradation system to enable real‑time, spatio‑temporal analysis and acute perturbation of Yap1 function in developing and regenerating tissues.
The work reports creation of an endogenous knock‑in line in medaka in which the Yap1 protein is fused to mGreenLantern (Yap1‑mGL) using CRISPR/Cas9 genome editing. This approach places a fluorescent reporter on the native Yap1 locus, allowing observation of endogenous expression levels and subcellular localisation without overexpression artifacts.
The source describes the generation and characterisation of this Yap1‑mGL knock‑in line as an enabling reagent for in vivo studies of Yap1 dynamics.
By virtue of the endogenous fluorescent tag, the Yap1‑mGL line permits four‑dimensional (3D across time) imaging of Yap1. The authors report the ability to resolve reporter fluorescence in real time and to monitor nuclear versus cytoplasmic localisation dynamics.
Importantly, the onset of reporter fluorescence was observed before gastrulation, indicating that the knock‑in permits tracking of Yap1 from very early embryonic stages. The system therefore supports longitudinal analyses spanning early development through later morphogenetic events.
Using the Yap1‑mGL reporter, the authors document tissue‑ and cell type–specific localisation patterns during somitogenesis. The data reveal differential nuclear and cytoplasmic localisation of Yap1‑mGL in distinct tissues and cell types as somites form.
These observations demonstrate the reporter’s capacity to capture context‑dependent subcellular distribution of endogenous Yap1 during a key morphogenetic window.
The medaka Yap1‑mGL line was applied to regenerative contexts. The authors report injury‑induced upregulation of Yap1‑mGL during larval spinal cord regeneration, showing that the reporter can detect dynamic changes in endogenous Yap1 abundance associated with tissue damage and repair.
This result highlights the utility of the knock‑in for studies that link mechanical or injury cues to Yap1‑dependent transcriptional responses in regenerative processes.
Beyond visualisation, the study couples the endogenous tag to a nanobody‑based degron system to achieve acute in vivo degradation of Yap1. Using this approach, Yap1 protein was degraded in living medaka, and this acute perturbation reproduced the phenotype observed in genetic loss‑of‑function mutants.
This demonstrates that nanobody‑mediated targeting of an endogenous fluorescently tagged protein can be used not only to visualise but also to functionally perturb the protein rapidly and reversibly in the organismal context.
Combining an endogenous fluorescent tag with a nanobody‑driven degron provides a twofold advantage: it enables precise spatio‑temporal visualisation of an endogenous signalling effector and permits acute, targeted perturbation of the same molecule in vivo. Applied to the conserved Hippo pathway effector Yap1, this strategy gives investigators a powerful platform to dissect how mechanical and chemical signals are integrated into transcriptional outputs during development and regeneration.
The approach may be broadly applicable to other signalling proteins where endogenous dynamics and acute functional manipulation are both required to resolve mechanism.
The source document is a bioRxiv preprint and has not undergone peer review. The abstract and summary provided key outcomes (knock‑in generation, early expression detection, tissue‑specific localisation, injury‑induced upregulation, and nanobody‑based degradation), but methodological details, quantitative data, controls, and extended results are not reported in the supplied source text. Those details would need to be consulted in the full manuscript or peer‑reviewed publication for experimental replication and critical evaluation.
The authors generated a CRISPR/Cas9‑mediated Yap1‑mGreenLantern knock‑in in medaka that enables 4D visualisation of endogenous Yap1 from pre‑gastrulation stages through somitogenesis and into larval regenerative responses. Coupling this endogenous tag to a nanobody‑based degron allowed acute degradation of Yap1 in vivo that recapitulated genetic loss‑of‑function phenotypes. Together, these tools provide a flexible platform to visualise and acutely perturb endogenous protein function in vivo, exemplified here for the Hippo pathway effector Yap1.