combining ilastik and flynotyper for accurate detection and quantification of ommatidia in drosophila melanogaster fly eye images

Unbiased Quantification of Ommatidia in Drosophila Eye Models

The Drosophila melanogaster genome contains ~75% of human disease-related gene orthologs. Additionally, during Drosophila eye development, approximately two-thirds of the genes in the genome are expressed, making the Drosophila eye an outstanding genetic system to investigate various molecular and cellular functions, development, and disease models1,2. Thus, the Drosophila eye system is a useful experimental tool to study various biological processes.
The Drosophila compound eye is a well-structured and comprehensive array of ~800 ommatidia that exhibit a symmetrical and hexagonal pattern3. The regularity of this hexagonal pattern can be used to estimate the effect of introducing mutations and gene expression changes in eye morphology4. Previous studies that require evaluation of eye morphology have heavily relied on manual ranking of the severity of eye phenotypes detected by the naked eye. To rank the eye phenotypes, external eye morphology images are taken by a stereomicroscope5,6. The eye phenotype of each group is assessed by splitting the external eye into four areas and calculating the proportion of degeneration in each area5,6. Then, the values are used to calculate averages that are compared with the values obtained from control flies7. The scoring is based on the extent of fusion, loss of ommatidia, and bristle organization7,8. Fly eye photos taken with a stereomicroscope are acquired by one researcher, and the eye phenotype analysis is performed by another researcher with triple validation sets7,8.
When it comes to ranking weak alterations in eye morphology with the naked eye, there are limitations4. To overcome these limitations, computational approaches such as FLEYE and Flynotyper have been developed1,9. Flynotyper is a novel computational method for quantitatively estimating morphological changes in the Drosophila eye system1. It automatically detects the Drosophila eye and individual ommatidium, calculating Phenotypic Scores (P-Scores) based on the irregularity of theeye1. A higher P-Score indicates the fly eye is more degenerated. This software was successfully used in quantifying the abnormality of Drosophila eyes10. Although Flynotyper ensures an automated process, it still cannot be successfully applied to some eye images taken by various light microscopy methods.
Qualitatively, we prefer a ring light source compared to a single-point light source, as it offers a more accurate representation of each ommatidium. However, when the ring light is used, it generates a ring-shaped shadow at the top of each ommatidium due to the hemispherical shape of the ommatidium. This ring-shaped shadow inhibits accurate ommatidial detection by Flynotyper, leading to incorrect calculation of P-Scores.
To overcome these issues, we implemented ilastik, a machine learning-based tool for various analyses, to classify ommatidia in fly eye images11. We then fed the ilastik-generated images into Flynotyper to calculate P-Scores. This allows us to quantify the Drosophila eye morphological defects unbiasedly1.

Author Spotlight: Quantifying Rough Eye Phenotypes in Drosophila Models of Amyotrophic Lateral Sclerosis with Frontotemporal Dementia

Quantifying Drosophila melanogaster Eye Phenotypes: A Computational Approach Integrating ilastik and Flynotyper

We're interested in developing disease models for diseases like ALS, FTD, and multisystem proteinopathy to understand their molecular pathologies. One model we use is the Drosophila eye system. The regularity and symmetrical pattern of the eye can be used to evaluate the effect of introducing mutations and gene expression changes.Our protocol addresses a gap in ways of quantifying rough eye phenotypes, specifically, the gap created by manual ranking. Manual ranking is based on fusion, loss of ommatidia, and bristle organization. However, it can lead to bias.Therefore, combining ilastik and Flynotyper creates for a more robust method for ranking abnormal eye phenotypes. Our protocol can be advantageous to manual ranking due to its robustness. Even non-trained laboratory members can perform successful quantification, whereas a more trained researcher is likely needed for manual ranking to avoid score-to-score differences between two different researchers.Therefore, this protocol can save time and resources. Our findings advance research because this protocol allows for more accurate quantification of rough eye phenotypes, especially weak alterations in eye morphology, Due to bias when manually ranking, combining ilastik and Flynotyper will lead to more accurate scoring, and therefore, result in more accurate published results.

Combining ilastik and Flynotyper for Accurate Detection and Quantification of Ommatidia in Drosophila melanogaster Fly Eye Images

To begin place the healthy and degenerated Drosophila fly eye images in the same folder. Ensure that the files are appropriately named to identify the experimental groups and distinguish between males and females. Open the Ilastik software.Click on Create New Project. Then from Other Workflows, select Cell Density Counting and select a location to save the project file. Sequentially, click on 1.Input Data, Add New, and Add Separate Images to choose the standard image. Then click on 2. Feature Selection and select the features.Choose more sigma values to increase sensitivity. Click on 3. Counting and set the foreground sigma value to 5.00.Using the foreground tool, mark 50 or more individual ommatidia on the standard image. Click on 3. Counting again and use the background tool to mark the area outside of the ommatidia.Draw green lines in a lattice shape around the ommatidia. Click on 4. Density Export and choose Export Image Settings to adjust image settings.Then click on Output File Info and select format tiff for further analysis in Flynotyper. Click on 5. Batch Processing, Select Raw Data Files, and select all experimental fly photos to be analyzed.The list of imported images to be analyzed is seen under Select Raw Data Files. Click Process All Files at the bottom of the 5. Batch Processing section.After the software completes processing, check the folder containing the experimental fly eye images. Confirm that black images named, your sample name probabilities, are present. In ImageJ, open the Ilastik-generated tiff file.Use the rectangle select tool to make a rectangle around the eye. Select Control X or Control C to crop the area and wait for a black box in the shape of the rectangle outline to appear. Sequentially, click File, New, and Internal Clipboard to open the cropped fly eye.To save the cut fly eye as a JPEG, click on File, Save As, and JPEG. In ImageJ, click on Plugins, and Flynotyper. Under Genotypes, select Add Genotypes and open the folder containing the cut fly eye images.Tick the Light Microscope and Vertically boxes. Then under Rank Ommatidia By, check Stability and Distance to the Center boxes. For the number of ranked ommatidia considered, set input as 200.Click Run and wait for the results of the analysis to appear. Copy and paste the sample file and P-score into statistical software for further analysis. Drosophila eye images processed with Ilastik and analyzed under ring light showed clearer ommatidial patterns compared to those without ring light enhancing the qualitative assessment.Flynotyper analysis indicated significantly higher phenotypic scores in the moderately and severely degenerated fly eyes compared to non-degenerated eyes. When Ilastik was not used, the P-scores between non-degenerated and moderately degenerated eyes were similar.

combining-ilastik-flynotyper-for-accurate-detection-quantification

Research

JoVE Journal

Developmental Biology

The Drosophila melanogaster compound eye is a well-structured and comprehensive array of around 800 ommatidia, exhibiting a symmetrical and hexagonal pattern. This regularity and ease of observation make the Drosophila eye system a powerful tool to model various human neurodegenerative diseases. However, ways of quantifying abnormal phenotypes, such as manual ranking of eye severity scores, have limitations, especially when ranking weak alterations in eye morphology. To overcome these limitations, computational approaches have been developed such as Flynotyper. The use of a ring light allows for better qualitative images accessing the intactness of individual ommatidia. However, these images cannot be analyzed by Flynotyper directly due to shadows on ommatidia introduced by the ring light. Here, we describe an unbiased way to quantify rough eye phenotypes observed in Drosophila disease models by combining two software, ilastik and Flynotyper. By preprocessing the images with ilastik, successful quantification of the rough eye phenotype can be achieved with Flynotyper.

The Drosophila eye system is a useful tool for studying various biological processes, particularly human neurodegenerative diseases. However, manual quantification of rough eye phenotypes can be biased and unreliable. Here we describe a method by which ilastik and Flynotyper are used to quantify eye phenotype in an unbiased way.

The Drosophila melanogaster compound eye is a well-structured and comprehensive array of around 800 ommatidia, exhibiting a symmetrical and hexagonal ...