Abstract
Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.
Highlights
Despite numerous and versatile genetic mosaic strategies available for genetically amenable model organism Drosophila melanogaster, none up to now was suited for efficient large-scale screening for cell non-autonomous effects on a combinations of genetic manipulations, non-autonomous effects are more challenging to investigate yet well known to play crucial roles in development and disease contexts such as cancer
The challenge applies to any tissue but is evident in the central nervous system (CNS) due to diversity of cell types and uniqueness of each lineage with respect to gene expression, size, projection patterns, as well as lethality frequently associated with their disruption
To illustrate in our specific case: no available genetic tool allowed large-scale screening for non-autonomous effects on neural tumor growth as animals harbouring neural tumors cannot be kept as a stable stock
Summary
Despite numerous and versatile genetic mosaic strategies available for genetically amenable model organism Drosophila melanogaster, none up to now was suited for efficient large-scale screening for cell non-autonomous effects on a combinations of genetic manipulations, non-autonomous effects are more challenging to investigate yet well known to play crucial roles in development and disease contexts such as cancer. A much needed, transformative, new tool would be: (i) a viable parental stock in which (ii) chosen individual lineages could be (iii) triggered to assume a deleterious genotype (iv) with temporal control (v) from which point they would become permanently labeled by a reporter and (vi) with which a single cross to existing stocks would produce progeny with genetically perturbed cell types of interest other than the labeled lineages. To illustrate in our specific case: no available genetic tool allowed large-scale screening for non-autonomous effects on neural tumor growth as animals harbouring neural tumors cannot be kept as a stable stock. Transcriptional activation systems include the yeast transcription factor GAL4 and its binding site, named Upstream Activating developmentally deleterious genotype
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