Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects.By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spa¨ tzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases

DeadEasy Caspase: Automatic Counting of Apoptotic Cells in Drosophila

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.This work was funded by Wellcome Trust Equipment Grant 073228 and EMBO YIP to AH, and by BBSRC and MRC studentships to JAP and ARL.Peer reviewe

Parameters that can be altered by the user and the consequences.

<p>This table sows the parameters that the user can modify after accessing through ImageJ theprogrammes written in Java. Any parameter modification will require cycles of validation and further modification by the user, until the desired accuracy for the sample and staining in use is reached.</p

Examples of application of DeadEasy Caspase to address biological questions.

<p>DeadEasy Caspase is used to count the number of apoptotic cells stained with Caspase in wild-type embryos at different stages and in H99 mutants lacking apoptosis. Numbers over box-plots indicate number of embryos analysed per genotype.</p

Properties of cells counted by DeadEasy Caspase.

<p>Properties of cells counted by DeadEasy Caspase.</p

DeadEasy Caspase: the mathematical algorithm.

<p>DeadEasy Caspase detects apoptotic cells in embryos stained with anti-cleaved-Caspase-3, and are characterised by low cytoplasmic signal, high background and volume ≥1.56 µm³. (A) Diagram showing the region of the embryonic VNC (blue) scanned for counting. (B) Caspase histogram. (C) Enlarged images to compare a raw image vs. the result (single confocal 0.25 µm slices shown). (D) Diagram of the algorithm. (E) Images showing the different steps of processing, correlating with each step in the diagram in (D). (F) Examples of bad quality stainings or images that must not be used for cell counting as they will produce false positives.</p

The extent of apoptosis in the Drosophila embryonic VNC.

<p>(A) A Drosophila embryonic VNC. The Region Of Interest (ROI) box in red indicates the areas analysed in (B,C). (B) 3D rendering of confocal stacks of sections through the VNC to show the abundant number of embryonic apoptotic cells stained with anti-cleaved-Caspase-3. (C) Cross section view of the image in (B).</p

Validation of DeadEasy Caspase.

<p>(A) During processing, DeadEasy creates a second stack of confocal images reproducing the identified objects in locations that correspond to those of cells in the original raw stack. By placing the mouse over each of the objects, an identifying number is revealed (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005441#pone-0005441-g002" target="_blank">Figure 2</a>), showing whether a cell is counted appropriately. We compared one cell at a time in this way through multiple stacks. (B) Using a second validation method, a merged stack can be created from the raw images (green) and the processed images with the identified objects (red). Colocalising cells in the merged stack (yellow) indicate the identified cells, green cells are false negatives and red cells are false positives.</p

How DeadEasy software works.

<p>How DeadEasy software works.</p