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Phenomics requires quantification of large volumes of image data, necessitating high throughput image processing approaches. Existing image processing pipelines for  Drosophila  wings, a powerful genetic model for studying the underlying genetics for a broad range of cellular and developmental processes, are limited in speed, precision, and functional versatility. To expand on the utility of the wing as a phenotypic screening system, we developed MAPPER, an automated machine learning-based pipeline that quantifies high-dimensional phenotypic signatures, with each dimension quantifying a unique morphological feature of the  Drosophila  wing. MAPPER magnifies the power of  Drosophila  phenomics by rapidly quantifying subtle phenotypic differences in sample populations. We benchmarked MAPPER’s accuracy and precision in replicating manual measurements to demonstrate its widespread utility. The morphological features extracted using MAPPER reveal variable sexual dimorphism across  Drosophila  species and unique underlying sex-specific differences in morphogen signaling in male and female wings. Moreover, the length of the proximal-distal axis across the species and sexes shows a conserved scaling relationship with respect to the wing size. In sum, MAPPER is an open-source tool for rapid, high-dimensional analysis of large imaging datasets. These high-content phenomic capabilities enable rigorous and systematic identification of genotype-to-phenotype relationships in a broad range of screening and drug testing applications and amplify the potential power of multimodal genomic approaches.

MAPPER: An Open-Source, High-Dimensional Image Analysis Pipeline Unmasks Differential Regulation of Drosophila Wing Features