A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling
- National Centre for Biological Sciences, India
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.
Reviewing Editor
- Jiwon Shim, Hanyang University, Republic of Korea
Version history
- Received: February 6, 2020
- Accepted: December 14, 2020
- Accepted Manuscript published: December 15, 2020 (version 1)
- Version of Record published: December 29, 2020 (version 2)
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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A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling
eLife 9:e55793.
https://doi.org/10.7554/eLife.55793
Further reading
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We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:
SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
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Human fetal development has been associated with brain health at later stages. It is unknown whether growth in utero, as indexed by birth weight (BW), relates consistently to lifespan brain characteristics and changes, and to what extent these influences are of a genetic or environmental nature. Here we show remarkably stable and lifelong positive associations between BW and cortical surface area and volume across and within developmental, aging and lifespan longitudinal samples (N = 5794, 4–82 y of age, w/386 monozygotic twins, followed for up to 8.3 y w/12,088 brain MRIs). In contrast, no consistent effect of BW on brain changes was observed. Partly environmental effects were indicated by analysis of twin BW discordance. In conclusion, the influence of prenatal growth on cortical topography is stable and reliable through the lifespan. This early-life factor appears to influence the brain by association of brain reserve, rather than brain maintenance. Thus, fetal influences appear omnipresent in the spacetime of the human brain throughout the human lifespan. Optimizing fetal growth may increase brain reserve for life, also in aging.
