Automatic cell counting in vivo in the larval nervous system of Drosophila

Abstract

Summary Identification and counting of cells is necessary to test biological hypotheses, for instance of nervous system formation, disease, degeneration, injury and regeneration, but manual counting is time consuming, tedious, and subject to bias. The fruit fly Drosophila is a widely used model organism to analyse gene function, and most research is carried out in the intact animal or in whole organs, rather than in cell culture. Inferences on gene function require that cell counts are known from these sample types. Image processing and pattern recognition techniques are appropriate tools to automate cell counting. However, counting cells in Drosophila is a complex task: variations in immunohistochemical markers and developmental stages result in images of very different properties, rendering it challenging to identify true cells. Here, we present a technique for counting automatically larval glial cells in three dimensions, from confocal microscopy serial optical sections. Local outlier thresholding and domes are combined to find the cells. Shape descriptors extracted from a data set are used to characterize cells and avoid oversegmentation. Morphological operators are employed to divide cells that could otherwise be missed. The method is accurate and very fast, and treats all samples equally and objectively, rendering all data comparable across specimens. Our method is also applicable to identify cells labelled with other nuclear markers and in sections of mouse tissues.

Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki

“…To ask what functions Kon might have in glia, as homozygous null kon mutants are lethal in the embryo, we tested the effects of kon knockdown using RNAi. The total number of Repo + glial cells in third-instar larval VNCs were counted in vivo automatically using DeadEasy Larval Glia software ( Forero et al, 2012 ). kon knockdown in all neurons ( elavGAL4>UASkonRNAi 106680 ) or only the Pros + NG ( alrmGAL4>konRNAi 106680 ), did not affect total glial cell number ( Fig.…”

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

“…The entire VNC was counted, using the edges of the optic lobes as anterior boundaries. Eve + cells in the larval CNS were counted automatically with DeadEasy Larval Glia software, which counts nuclear stains (see previous section; Forero et al, 2012 ). For Eve + cell counting, the thoracic (T1–T3) and posterior tip cells were excluded because cells there are too packed together, and only the cells from abdominal segments A1–A6 were counted.…”

Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila

“…For automatic whole-brain counting of Histone::YFP-positive cells, driven by alrm-GAL4 or NP6520, the ImageJ/FIJI plug-in DeadEasy Larval Glia (Forero et al, 2012) was used. The region of interest (ROI) was defined as a hemisphere of the region from the area around the brain neuropil to anterior of the group of exit glial cells in the first thoracic segment.…”