Abstract
BACKGROUND: Although some aspects of limb development can be treated as a 2D problem, a true understanding of the morphogenesis and patterning requires 3D analysis. Since the data on gene expression patterns are largely static 3D image stacks, a major challenge is an efficient pipeline for staging each data-set, and then aligning and warping the data into a standard atlas for convenient visualisation. RESULTS: We present a novel bioinformatic pipeline tailored for 3D visualization and analysis of developing limb buds. The pipeline integrates key steps such as data acquisition, volume cleaning, surface extraction, staging, alignment, and advanced visualization techniques. Its modular design allows researchers to customize workflows while maintaining compatibility with tools such as Fiji and Vedo. The pipeline can be accessed at https://github.com/LauAvinyo/limblab . CONCLUSIONS: The pipeline advances 3D gene expression analysis in limb development by integrating flexible tools for staging, alignment, and visualization. It is user-friendly, scalable to other samples, and optimized for research needs. Future updates will enhance customization and expand applicability to other species and developmental biology fields.