A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling
Abstract
Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.
Data availability
Full genome sequencing for isogenized Attp40 Stock submitted to NCBI (BioProject ID PRJNA606147). Full genome sequencing for S2R+ cells submitted to NCBI (Bioproject ID PRJNA606149). Images for PI signaling genetic screen saved at Open Source Frame https://osf.io/pt7zu/?view_only=14642fc3a5d74e408fb3766c2555393f
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Full genome sequencing for isogenized Attp40 StockNCBI, BioProject ID PRJNA606147.
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Full genome sequencing for S2R+ cellsNCBI, BioProject ID PRJNA606149.
Article and author information
Author details
Funding
Department of Biotechnology, Ministry of Science and Technology, India (BT/PRJ3748/GET/l 19/27/2015)
- Deepti Trivedi
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Padinjat Raghu
Wellcome-DBT India Alliance (IA/S/14/2/501540)
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Bishal Basak
- Padinjat Raghu
National Centre for Biological Sciences (core)
- Deepti Trivedi
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Awadhesh Pandit
- Bishal Basak
- Padinjat Raghu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Trivedi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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Stem cell self-renewal often relies on asymmetric fate determination governed by niche signals and/or cell-intrinsic factors but how these regulatory mechanisms cooperate to promote asymmetric fate decision remains poorly understood. In adult Drosophila midgut, asymmetric Notch (N) signaling inhibits intestinal stem cell (ISC) self-renewal by promoting ISC differentiation into enteroblast (EB). We have previously shown that epithelium-derived Bone Morphogenetic Protein (BMP) promotes ISC self-renewal by antagonizing N pathway activity (Tian and Jiang, 2014). Here, we show that loss of BMP signaling results in ectopic N pathway activity even when the N ligand Delta (Dl) is depleted, and that the N inhibitor Numb acts in parallel with BMP signaling to ensure a robust ISC self-renewal program. Although Numb is asymmetrically segregated in about 80% of dividing ISCs, its activity is largely dispensable for ISC fate determination under normal homeostasis. However, Numb becomes crucial for ISC self-renewal when BMP signaling is compromised. Whereas neither Mad RNA interference nor its hypomorphic mutation led to ISC loss, inactivation of Numb in these backgrounds resulted in stem cell loss due to precocious ISC-to-EB differentiation. Furthermore, we find that numb mutations resulted in stem cell loss during midgut regeneration in response to epithelial damage that causes fluctuation in BMP pathway activity, suggesting that the asymmetrical segregation of Numb into the future ISC may provide a fail-save mechanism for ISC self-renewal by offsetting BMP pathway fluctuation, which is important for ISC maintenance in regenerative guts.