Abstract
The Drosophila egg chamber, whose development is divided into 14 stages, is a well-established model for developmental biology. However, visual stage determination can be a tedious, subjective and time-consuming task prone to errors. Our study presents an objective, reliable and repeatable automated method for quantifying cell features and classifying egg chamber stages based on DAPI images. The proposed approach is composed of two steps: 1) a feature extraction step and 2) a statistical modeling step. The egg chamber features used are egg chamber size, oocyte size, egg chamber ratio and distribution of follicle cells. Methods for determining the on-site of the polytene stage and centripetal migration are also discussed. The statistical model uses linear and ordinal regression to explore the stage-feature relationships and classify egg chamber stages. Combined with machine learning, our method has great potential to enable discovery of hidden developmental mechanisms.
Highlights
The development of the egg chamber during Drosophila oogenesis is divided into 14 stages[1]
Particular stages were initially assigned to egg chambers by skilled biologists in our team based on standard descriptive guidance[1] (Fig. 1)
The development of an organism is often accompanied by increase in size, and this is the case in Drosophila egg chamber stage development
Summary
The development of the egg chamber during Drosophila oogenesis is divided into 14 stages[1]. Even an expert sometimes has difficulty assigning correct stages to inter-stage transitional egg chambers. To address these problems, and to reduce the bias caused by human perceptual variation, an objective, reliable and standardized egg chamber stage identification method is greatly needed. We developed a toolbox that applies understanding of egg chamber morphology[1] extracted automatically from DAPI staining to determine egg chamber stages. We collected 172 confocal microscopy images from different stages to train and evaluate our automatic stage identification method. We applied our method to successfully confirm the occasional appearance of Broad expression as early as stage 5, and unambiguously demonstrated that egg chambers with germline Delta mutation entered midoogenesis, even though the follicle cells did not show the appropriate biomarker, Br 5. Our findings clarified our understanding that stage 6 should be considered as an entering stage of endocycle, and stage 7 completes endocycle entry[2,3,4,5,6,9,10]
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