Abstract
Development, cancer, neurodegenerative and demyelinating diseases, injury, and stem cell manipulations are characterised by alterations in cell number. Research into development, disease, and the effects of drugs require cell number counts. These are generally indirect estimates, because counting cells in an animal or organ is paradoxically difficult, as well as being tedious and unmanageable. Drosophila is a powerful model organism used to investigate the genetic bases of development and disease. There are Drosophila models for multiple neurodegenerative diseases, characterised by an increase in cell death. However, a fast, reliable, and accurate way to count the number of dying cells in vivo is not available. Here, we present a method based on image filtering and mathematical morphology techniques, to count automatically the number of dying cells in intact fruit-fly embryos. We call the resulting programme DeadEasy Caspase. It has been validated for Drosophila and we present examples of its power to address biological questions. Quantification is automatic, accurate, objective, and very fast. DeadEasy Caspase will be freely available as an ImageJ plug-in, and it can be modified for use in other sample types. It is of interest to the Drosophila and wider biomedical communities. DeadEasy Caspase is a powerful tool for the analysis of cell survival and cell death in development and in disease, such as neurodegenerative diseases and ageing. Combined with the power of Drosophila genetics, DeadEasy expands the tools that enable the use of Drosophila to analyse gene function, model disease and test drugs in the intact nervous system and whole animal.
Highlights
The quantitative analysis of cell death is required to solve fundamental questions of developmental biology and to understand disease
Drosophila is a very powerful model organism that has led to the discovery of gene networks and gene functions involved in development, cancer and neurodegeneration [1]
Approaches that estimate cell number based on general properties miss phenotypes in cell number that are subtle, that do not lead to anatomically visible consequences or that affect a fraction of cells
Summary
The quantitative analysis of cell death (apoptosis) is required to solve fundamental questions of developmental biology and to understand disease. Functional analyses of molecules controlling cell number have frequently been carried out in cell culture or after cell dissociation; counting cells in the intact animal (i.e. in vivo) is generally carried out manually, and may consist of estimates of number of cells stained with a particular cell marker or inferences from anatomical alterations [2,3,4,5]. In Drosophila, manual counting, throughout the nervous system, is carried out using antibodies to visualise particular cell states or cell lineages [6,7,8,9,10,11] While these methods are most accurate and most appropriate for certain questions, they can be extremely time-consuming and/or inappropriate for other questions. DeadEasy Caspase has been written in Java as a freely available ImageJ plug-in, and the programme is accessible for further creative modifications by researchers beyond the Drosophila community and within the general biomedical community
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